Application of sesquiterpene lactone compound in preparation of medicine for treating triple negative breast cancer

Abstract

The invention discloses application of a sesquiterpene lactone compound in preparation of a medicine for treating triple-negative breast cancer, an elephantopus tomentosus monomer EM-2 shown in a formula (I) is a novel sesquiterpene lactone compound extracted and identified from elephantopus tomentosus, and has a good proliferation inhibition effect on cisplatin drug-resistant cells of the triple-negative breast cancer. The compound has relatively low toxicity to liver epithelial LO2 cells and mammary epithelial cells MCF-10A; on the other hand, EM-2 improves the sensitivity of triple negative breast cancer cells to cis-platinum and reverses drug resistance by blocking an AKT pathway activated by cis-platinum and autophagy. In addition, it is found for the first time that the combined use of EM-2 and cis-platinum has a remarkable curative effect in treatment of triple negative breast cancer, and the application prospect is wide.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to the application of sesquiterpene lactone compounds in the preparation of drugs for treating triple-negative breast cancer. Background Art

[0002] Triple-negative breast cancer (TNBC) is a highly invasive subtype of breast cancer, with immunohistochemical staining of estrogen receptor (ER) and progesterone receptor (PR) less than 1% and lack of overexpression and / or gene amplification of human epidermal growth factor receptor 2 (HER-2). TNBC accounts for about 15% to 20% of all breast cancers, and is characterized by a young age of onset, family history, high heterogeneity, early local recurrence and distant metastasis. Due to the lack of effective targeted therapy, adjuvant therapy for TNBC is still mainly chemotherapy. In advanced metastatic triple-negative breast cancer, the median survival of current treatment options is only 13-18 months. Although emerging immunotherapy and anti-angiogenic drugs have been used in the treatment of TNBC in recent years, their efficacy is minimal and has not significantly improved the survival of TNBC patients. They are mainly used for rescue treatment of patients with recurrence and metastasis.

[0003] Platinum drugs are commonly used in triple-negative breast cancer chemotherapy. They have strong anti-tumor effects and definite efficacy. When used in combination with anthracyclines and / or taxanes, they can significantly improve the pathological complete remission rate (pCR) in the treatment of TNBC and locally advanced breast cancer. However, the drug resistance of platinum drugs limits their clinical application in the treatment of breast cancer.

[0004] On the other hand, although platinum plays an important role in the treatment of triple-negative breast cancer, its clinical efficacy is limited by side effects, primary and acquired treatment resistance. There are many factors that lead to DDP resistance, such as activation of drug efflux systems, increased biotransformation and detoxification in the liver, increased DNA damage repair, hypoxia in the tumor microenvironment, defects in cell death pathways, and increased pro-survival mechanisms, and the problem of DDP chemotherapy resistance has not yet been solved clinically. Summary of the invention

[0005] The present application aims to provide the use of sesquiterpene lactone compounds in the preparation of drugs for treating triple-negative breast cancer.

[0006] The technical solution adopted by the present invention is:

[0007] In the first aspect of the present application, a sesquiterpene lactone compound is provided for use in the preparation of a drug for treating or adjuvant treating triple-negative breast cancer, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

[0008]

[0009] The second aspect of the present application provides the use of a sesquiterpene lactone compound in the preparation of a drug for inhibiting the proliferation of triple-negative breast cancer cells, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof.

[0010] The third aspect of the present application provides the use of sesquiterpene lactone compounds in the preparation of compounds for blocking cisplatin-activated AKT pathway and enhancing autophagy, wherein the sesquiterpene lactone compounds are compounds represented by formula (I), pharmaceutically acceptable salts thereof or stereoisomers thereof or prodrug molecules thereof.

[0011] AKT and protective autophagy are both survival pathways activated by tumor cells when they are starved or stressed. Inhibition of AKT and / or inhibition of autophagy can promote tumor cell death. It is generally believed that AKT can inhibit autophagy after activation. AKT phosphorylates ULK1 (S758) by activating mTOR, inhibiting the activity of ULK1 protein kinase complex and thus inhibiting autophagy. In addition, AKT can also mediate the phosphorylation of Beclin 1 (S234 and S295) sites, promoting the formation of Beclin 1 / 14-3-3 / vimentin intermediate filament complexes and thus inhibiting autophagy.

[0012] However, a few researchers have found that inhibiting the AKT pathway can also inhibit autophagy. For example, the CD13 inhibitor Ubenimex inhibits autophagy by inhibiting the PI3K / AKT / NF-κB pathway, reversing the DDP resistance of gastric cancer cells. 4-Acetylantroquinonol B significantly inhibits the expression of Atg5 and Atg7 by inhibiting the PI3K / AKT / mTOR / p70S6K signaling pathway, while reducing autophagy flux, thereby increasing the DDP sensitivity of ovarian cancer.

[0013] The fourth aspect of the present application provides the use of a sesquiterpene lactone compound in the preparation of an anti-triple-negative breast cancer synergist, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof.

[0014] The fifth aspect of the present application is the use of sesquiterpene lactone compounds in the preparation of resistance reversal agents for triple-negative breast cancer, wherein the sesquiterpene lactone compounds are compounds represented by formula (I), pharmaceutically acceptable salts thereof or stereoisomers thereof or prodrug molecules thereof.

[0015] The sixth aspect of the present application provides the use of a sesquiterpene lactone compound in combination with cisplatin in the preparation of a drug for the treatment or adjuvant treatment of triple-negative breast cancer, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof.

[0016] The present application also proposes a pharmaceutical composition for treating triple-negative breast cancer, comprising: a sesquiterpene lactone compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof.

[0017] The "pharmaceutically acceptable salt" is a conventional non-toxic salt formed by the reaction of the compound of general formula (I) with an inorganic acid or an organic acid. For example, the conventional non-toxic salt can be prepared by the reaction of the compound of general formula (I) with an inorganic acid or an organic acid, wherein the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid and phosphoric acid, and the organic acid includes citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionylsulfonic acid. Acid, etc.; or the compound of the general formula (I) forms an ester with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid, and then forms a sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt with an inorganic base; or the compound of the general formula (I) forms a methylamine salt, ethylamine salt or ethanolamine salt with an organic base; or the compound of the general formula (I) forms an ester with lysine, arginine, or ornithine, and then forms a corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, or phosphoric acid, or a corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid and ethanesulfonic acid.

[0018] More specifically, in the above technical solution, the dosage form is selected from: injection, tablet, capsule, kit or patch. Those skilled in the art can prepare the above pharmaceutical composition into various dosage forms according to specific circumstances. The preparation method is a well-known technology in the art, so this application will not be repeated.

[0019] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0020] 1. The inventors found through MTT, cell cloning and EdU experiments that EM-2 has a good anti-tumor effect on human triple-negative breast cancer MDA-MB-231 parental and cisplatin-resistant cells, and has low toxicity to normal epithelial cells. The IC50 for human triple-negative breast cancer MDA-MB-231 / DDP and MDA-MB-231 cells are 18.43±0.60 and 11.28±0.62μM, respectively, while the IC50 for human normal breast epithelial cells MCF-10A and human normal liver epithelial cells LO2 are 23.48±1.45 and 34.63±3.97μM, respectively. And EM2 promotes apoptosis of triple-negative breast cancer MDA-MB-231 parental and cisplatin-resistant cells in a concentration-dependent manner.

[0021] 2. EM-2 can inhibit the AKT pathway and block cellular autophagy in human triple-negative breast cancer MDA-MB-231 parental and cisplatin-resistant cells.

[0022] 3. The inventors found through MTT experiments that after DDP was combined with low doses (between IC20 and IC30) of EM-2, the IC50 of DDP against human triple-negative breast cancer MDA-MB-231 parental and cisplatin-resistant cells decreased from 20.09±1.06 and 108.4±6.01 μM to 5.73±0.29 and 4.64±0.04 μM, respectively. The combination of EM-2 and cisplatin can promote apoptosis of human triple-negative breast cancer MDA-MB-231 parental and cisplatin-resistant cells.

[0023] 4.EM-2 increases the sensitivity of triple-negative breast cancer cells to cisplatin and reverses drug resistance by blocking the AKT pathway and autophagy activated by cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 There is activation of AKT pathway and autophagy in triple-negative breast cancer MDA-MB-231 / DDP-resistant cells;

[0026] Figure 2 DDP activates the AKT pathway and autophagy in MDA-MB-231 parental and DDP-resistant cells;

[0027] Figure 3 Inhibition of AKT pathway can increase the sensitivity of MDA-MB-231 / DDP cells to DDP;

[0028] Figure 4 Silencing AKT can increase the sensitivity of MDA-MB-231 / DDP cells to DDP;

[0029] Figure 5 To block autophagy, the sensitivity of MDA-MB-231 / DDP cells to DDP was increased;

[0030] Figure 6 The anti-breast cancer and pro-apoptotic effects of EM-2;

[0031] Figure 7 EM-2 is a dual inhibitor of AKT and autophagy;

[0032] Figure 8 EM-2 increases the sensitivity of MDA-MB-231 cells to DDP and reverses the DDP resistance of MDA-MB-231 / DDP cells;

[0033] Fig. 9 EM-2 increases the sensitivity of MDA-MB-231 cells to DDP and reverses the DDP resistance of MDA-MB-231 / DDP cells through dual inhibition of AKT pathway and autophagy. DETAILED DESCRIPTION

[0034] In order to more clearly understand the technical contents of the present invention, the following examples are specifically described in detail in conjunction with the accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0035] EM2 is a sesquiterpene lactone compound extracted from the traditional Chinese herbal medicine Euphorbia pulegium, named 2β-methoxy-2-deethoxyphantomolin, with a molecular formula of C 20 H 24 O6, molecular weight 360, was isolated and purified by the School of Pharmacy of Jinan University, and its structure is shown below (I). EM2 powder was dissolved in DMSO to a 40 mM mother solution, packaged and stored at -80 °C

[0036]

[0037] 1. Materials and Methods

[0038] Cell lines: Human breast cancer cells MDA-MB-231, human normal breast epithelial cells MCF-10A and human normal liver epithelial cells LO2 were purchased from the American Type Culture Collection (ATCC). Human breast cancer cisplatin-resistant cells MDA-MB-231 / DDP were prepared by Sun Yat-sen Memorial Hospital, Sun Yat-sen University.

[0039] Methods: MTT assay was used to detect the toxicity of EM2 on normal human epithelial cells. MTT, cell clone formation and EdU assays were used to detect the effects of EM2 alone or EM2 combined with cisplatin on the proliferation of triple-negative breast cancer cells. MTT, cell clone formation and EdU assays were used to detect the sensitivity of triple-negative breast cancer cisplatin-resistant cells to cisplatin after inhibiting AKT or blocking autophagy. Immunoblotting was used to detect the effects of EM2 alone or EM2 combined with cisplatin on apoptosis, AKT and autophagy pathways. Immunofluorescence was used to detect the changes in p62 fluorescence. Annexin-FITC / PI double staining was used to detect the effects of EM2 alone or EM2 combined with cisplatin on apoptosis. qRT-PCR was used to detect the effects of AKT and autophagy-related genes.

[0040] MTT experiment: (1) Experimental design: zero adjustment group, control group and drug addition group. The zero adjustment group only added culture medium, the control group added an equal volume of DMSO, and the drug addition group added culture medium containing cells and experimental drugs. Five replicate wells were set for each group.

[0041] (2) When the cells are cultured to the logarithmic growth phase, digest the cells with trypsin digestion solution containing 0.25% EDTA, and terminate the digestion with culture solution containing 10% FBS and blow into a single cell suspension. Then collect the cells by low-speed centrifugation at 800rpm for 3min. Calculate the cell density after resuspending the cells in culture solution. At a density of 4000-6000 cells per well, seed 100μL of cell suspension into a 96-well plate, and continue to culture overnight. Fill the periphery of the well plate with 200μL 1×PBS to reduce evaporation of the culture solution.

[0042] (3) Use the doubling dilution method to dilute the drug to twice the experimental concentration. Add 100 μL of the drug to the cells to dilute the drug concentration to one-fold to the experimental working concentration. After 48 hours of culture, add 20 μL of MTT solution (5 mg / mL) to each well of the zero adjustment group, control group, and experimental group, and then continue to culture for 4-6 hours. After absorbing the mixture of culture medium and MTT, add 100 μL of DMSO solution to each well and shake at a low speed for 10 minutes. After the formazan blue-purple crystals are fully dissolved, use a multifunctional microplate reader to detect the absorbance at 570 nm, and calculate the survival rate of the cells in each group by calculating the average absorbance value of each group.

[0043] (4) Cell survival rate (%) = (average absorbance value of experimental group - average absorbance value of zero-adjustment group) / (average absorbance value of control group - average absorbance value of zero-adjustment group) × 100%. IC50 (Mean±SD) was calculated and plotted using GraphPad Prism 7.0.

[0044] Cell cloning experiment: (1) Take cells in the logarithmic growth phase, digest the cells with trypsin digestion solution containing 0.25% EDTA, centrifuge at low speed, remove the supernatant, and resuspend in culture medium containing 10% FBS. After counting the cells, the cells are evenly seeded into a six-well plate with about 500-1000 cells per well. Gently shake the 6-well plate, observe under a microscope to ensure that the cells are evenly seeded in the six-well plate, and then put it into a cell culture incubator.

[0045] (2) After the cells adhere to the wall, remove the supernatant and add 2 mL of culture medium containing different concentrations of drugs to each well to treat the cells according to the set experimental plan. Then place the six-well plate in a cell culture incubator and culture it for 7 to 14 days.

[0046] (3) After the culture is completed, discard the supernatant culture medium. Slowly add 1 mL of 1× PBS into the wells along the wall of the 6-well plate and gently shake to wash away the residual supernatant culture medium. Be careful not to blow the cells with a pipette to prevent the adherent cells from being washed off. This washing process is performed twice. Then add 1 mL of 4% paraformaldehyde solution to each well to fix the cells for 30 minutes to 1 hour.

[0047] (4) After the treatment time is reached, remove the culture medium, rinse gently with 1×PBS 2-3 times, add 1mL of 4% paraformaldehyde solution, and fix the cells for 20 minutes. Discard the fixative, gently shake and wash with 1×PBS 1-2 times, then add 1mL of 0.1% crystal violet staining solution and stain the cells for 30 minutes. Aspirate the 0.1% crystal violet staining solution, add tap water to wash 2-3 times, invert and dry, take pictures on the film observation light, record the clone formation, and record the tightly packed cell colony with more than 50 cells as one clone.

[0048] (5) Add 1 mL of 30% glacial acetic acid solution to each well and shake at low speed for 10 min. After the cell clones are fully dissolved, use a multifunctional microplate reader to detect the absorbance at 450 nm, calculate the average absorbance value of each group, and use GraphPad Prism 7.0 to draw a graph.

[0049] EdU experiment: (1) When the cells are cultured to the logarithmic growth phase, digest them and collect the cells by centrifugation. After counting, 100 μL of the cell suspension is seeded into a 96-well plate at a density of 5,000 cells per well. Set up 3 replicate wells for each group and continue culturing overnight.

[0050] (2) After the cells adhered, 100 μL of the corresponding drug was added to each well and cultured for another 24 h.

[0051] (3) After aspirating the culture medium, add 100 μL of fresh culture medium containing 10 μM EdU working solution and continue incubation for 2.5 h.

[0052] (4) After absorbing the EdU labeling solution, add 100 μL of 4% paraformaldehyde to fix for 20 min. After removing the fixative, add 100 μL of 2 μg / μL glycine solution and continue incubating at room temperature for 5 min to remove the residual 4% paraformaldehyde.

[0053] (5) Remove the glycine solution, add 100 μL of 3% BSA solution, wash 1-2 times, then add 100 μL of 0.5% Triton X-100 and incubate at room temperature for 15 min.

[0054] (6) Aspirate 0.5% Triton X-100, wash the cells 1-2 times with 3% BSA, then add 100 μL of Click-iT reaction mixture prepared according to the reagent instructions and incubate at room temperature in the dark for 30 min.

[0055] (7) Remove the reaction mixture, add 100 μL of 3% BSA solution to each well for washing 1-2 times, then add 100 μL of 5 μg / mL Hochest 33342 solution, incubate at room temperature in the dark for 15 min, then wash 1-2 times with 100 μL of 3% BSA solution, and add 50 μL of anti-fluorescence quencher.

[0056] (8) Take photos and analyze under an inverted fluorescence microscope. kFluor488: Ex / Em = 495nm / 519nm, green fluorescence. Hoechst 33342 (nuclear DNA dye): Ex / Em = 350nm / 461nm, blue fluorescence.

[0057] qRT-PCR experiments:

[0058] (1) Total RNA extraction - TRIzol method

[0059] Wear masks, gloves and head covers throughout the experiment and use enzyme-free pipette tips and consumable reagents to avoid RNase contamination.

[0060] 1) Take cells in the logarithmic growth phase, 2.0×10 6 The cells were inoculated into a 6-well plate at a density of 100 μg / mL and placed in an incubator overnight to adhere to the plate. The original culture medium was removed and 2 mL of the culture medium containing the drug was added to each well and cultured for 24 h.

[0061] 2) After the drug acts on the cells, remove the culture supernatant and wash twice with pre-cooled 1× PBS.

[0062] 3) Add 1 mL of TRIzol to each well to lyse the cells. After standing on ice for 5 minutes, transfer to a 1.5 mL enzyme-free centrifuge tube and pre-cool in a high-speed refrigerated centrifuge.

[0063] 4) Add 200 μL of chloroform at a ratio of TRIzol:chloroform = 5:1. After vigorous vortexing for 15 seconds, let it stand for 3 minutes. After observing the liquid phase separation, use a high-speed refrigerated centrifuge at 4°C, 12,000 rpm for 15 minutes.

[0064] 5) After centrifugation, transfer 200-400 μL of the upper aqueous phase to a new enzyme-free microcentrifuge tube, add an equal amount of isopropanol at a 1:1 ratio, and continue to use a high-speed refrigerated centrifuge at 4°C, 12,000 rpm, and centrifuge for 10 minutes.

[0065] 6) Discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water to wash the precipitate, centrifuge at 4°C, 7500 rpm for 5 min, and wash twice.

[0066] 7) Absorb all 75% ethanol, open the cap of the centrifuge tube, and invert it at room temperature to allow the 75% ethanol to evaporate completely. When the white precipitate is about to become transparent, add enzyme-free water to dissolve the RNA precipitate. Use NanoDrop 2000 micro-volume UV spectrophotometer to detect the concentration and purity of RNA, and then store it in a -80°C refrigerator.

[0067] (2) Reverse transcription-PCR (RT-PCR)

[0068] Table 1 Reverse transcription PCR related reagents and dosage

[0069]

[0070] After mixing, incubate at 37℃ for 15min, incubate at 85℃ for 5sec, and store the samples at -20℃.

[0071] (3) Real-time fluorescence quantitative PCR

[0072] 1) Premix system: 20 μL. Three replicate wells are set for each gene detected in each treatment sample, and the cDNA template comes from the product of the reversal system.

[0073] Table 2 Real-time fluorescence quantitative PCR related reagents and dosage

[0074]

[0075] 2) qPCR procedure: three-step method

[0076] Pre-denaturation: 95℃10min; Amplification (40 cycles): Denaturation: 95℃15sec, Annealing: 60℃30sec, Extension: 72℃30sec; Melting curve program uses the instrument default settings. -ΔΔCt Methods Relative gene expression levels were calculated.

[0077] Table 3 Primer sequences in qPCR program

[0078]

[0079]

[0080] Annexin V-FITC / PI double staining experiment: (1) Digest, resuspend and count the cells in the logarithmic growth phase at 2.0×10 5 Cells were inoculated into 6-well plates, and 1 mL of culture medium was added to each well.

[0081] (2) After the cells adhere to the wall, the supernatant is removed and the cells are treated with drugs at different concentrations for 24 h or 48 h according to experimental requirements, so that the total volume of the culture medium containing different concentrations of drugs in each well is 2 mL.

[0082] (3) Collect cells by digestion with EDTA-free trypsin to avoid false positives caused by the combination of EDTA and Annexin V. After washing the cells twice with pre-cooled 1× PBS, add 500 μL of Bind Buffer to the sample to resuspend the cells, then add 5 μL of Annexin V-FITC and 5 μL of PI staining solution, mix well, and incubate at room temperature in the dark for 15-20 minutes. Then, use flow cytometry to detect apoptosis of the samples.

[0083] Western blot experiment: (1) Take 2.0×10 6 The cells were seeded in 6-well plates and cultured overnight. Different concentrations of drugs were then added for 24 h according to experimental needs.

[0084] After drug treatment, remove the culture medium, add pre-cooled 1×PBS to rinse, and then add 100-200μL RIPA strong lysis buffer on ice, in which the protease inhibitor PMSF and protein phosphatase inhibitor mixture are pre-added. After lysis on ice for half an hour, collect the cell lysate into an enzyme-free centrifuge tube. Turn on the high-speed refrigerated centrifuge in advance and separate the protein supernatant by centrifugation at 4℃, 12000rpm for 15min. Transfer the protein supernatant to a new enzyme-free centrifuge tube and determine the protein concentration using the BCA kit.

[0085] After protein quantification, add SDS-PAGE protein loading buffer (5×) to dilute to 1×, heat in a 100°C metal bath for 10 min, and store the protein supernatant in aliquots at -80°C.

[0086] (2) Prepare polyacrylamide gel of appropriate concentration according to the molecular weight of the target protein to be detected. After assembling the electrophoresis device, add 1× electrophoresis working solution. Then load the rainbow protein marker and sample protein for electrophoresis. After electrophoresis at 80V for 30 minutes, switch to 120V and continue electrophoresis until the rainbow protein marker of the target protein molecular weight runs away completely.

[0087] (3) After the electrophoresis is completed, the excess gel is removed and laid flat on the transfer filter paper. The 0.22μM PVDF membrane is activated by soaking in methanol before use. After bonding with the glue, remove the bubbles and then insert it into the transfer clip, with the glue side facing the black side of the transfer clip. The black side of the transfer clip is facing the negative pole of the transfer tank, and the white side is facing the positive pole of the transfer tank. After filling with transfer solution, transfer the membrane with a constant current of 150mA for 90 minutes, and fill the periphery of the transfer tank with ice cubes.

[0088] (4) After the transfer, soak the membrane in 5% blocking milk, shake slowly, and incubate at room temperature for 1.5 hours.

[0089] (5) After blocking, wash off excess milk particles with 1×TBS buffer. Dilute the primary antibody with the primary antibody diluent at 1:1000, then soak the membrane in the primary antibody and incubate overnight at 4°C. After incubation with the primary antibody, recover the primary antibody and add 1×TBST membrane wash solution. Set the shaker at 90 rpm for 10 min each time, for a total of 3 times. Dilute the secondary antibody with 5% skim milk at a ratio of 1:7000, soak the PVDF membrane completely in the secondary antibody, and incubate at room temperature for 1 hour. After incubation, add 1×TBST membrane wash solution, set the shaker at 90 rpm for 10 min each time, for a total of 3 times. After washing the membrane, apply the special sensitive ECL chemiluminescent solution on the membrane, expose it with a chemiluminescent imager, and collect and analyze the experimental results.

[0090] Table 4 Antibodies related to immunoblotting and their suppliers

[0091]

[0092]

[0093] siRNA experiment: (1) Take 8×10 cells in logarithmic growth phase 5 The cells in each well were inoculated into a 6-well plate at a density of 1.5×10.5, cultured with complete culture medium without double antibodies, and placed in an incubator to continue culturing overnight to adhere to the wall.

[0094] (2) Dilute the si RNA solution and the transfection reagent according to the instructions of the transfection reagent. Gently mix the diluted si RNA solution and the transfection reagent and place them at room temperature for 15 minutes.

[0095] (3) Aspirate the complete culture medium without dual antibodies in the cell culture and rinse the cells twice with PBS.

[0096] (4) Mix the transfection complex with the complete culture medium without dual antibodies, add it to the well plate, and gently shake the 6-well plate to evenly disperse the mixture. At the same time, the treatment group is treated with drugs according to the experimental plan.

[0097] (5) Continue culturing in a CO2 incubator for 48 h before conducting subsequent experiments.

[0098] Immunofluorescence experiment: (1) Take cells in the logarithmic growth phase and inoculate them in a 96-well plate at a density of 5,000 cells per well. Set up 3 replicate wells for each group and continue to culture them in an incubator overnight to adhere to the plate.

[0099] (2) After the cells adhered, add drugs of corresponding concentration to each well and continue culturing for 24 h.

[0100] (3) After removing the culture medium, wash twice with pre-cooled 1× PBS, add 100 μL of 4% paraformaldehyde and fix the cells for 20 min.

[0101] (4) Remove 4% paraformaldehyde, wash twice with pre-cooled 1× PBS, then add 0.1% Triton-X and incubate at room temperature for 10 min.

[0102] (5) Add 10% goat serum to just cover the cells and block at 37°C for 1 h.

[0103] (6) Remove 10% goat serum and wash with pre-cooled 1× PBS. Dilute the primary antibody to a working solution at a ratio of 1:200 using 5% BSA. Incubate at 4°C overnight. Rinse the cells twice with 1× PBS containing 2% Tween 20.

[0104] (7) Add the corresponding fluorescent secondary antibody until it just covers the cells, dilute it at a ratio of 1:100, and incubate at room temperature for 1 hour. Aspirate the secondary antibody and wash twice with 1× PBS containing 2% Tween 20.

[0105] (8) Add 100 μL of ready-to-use DAPI staining solution to each well and incubate at room temperature in the dark for 10 min.

[0106] (9) Aspirate DAPI, wash twice with pre-cooled 1× PBS, add anti-fluorescence quencher, and observe under an inverted fluorescence microscope.

[0107] Statistics and analysis: SPSS Statistics 26 and GraphPad Psism 8.0 were used for data processing and analysis. One-way ANOVA was used to test the differences between groups of more than two samples. P < 0.05 was considered statistically significant.

[0108] Example 1

[0109] In order to study whether there is AKT pathway activation and autophagy activation in triple-negative breast cancer MDA-MB-231 / DDP resistant cells, the inventors used MTT assay to detect the half-inhibitory concentration IC50 (48h) of MDA-MB-231 and MDA-MB-231 / DDP cells for cisplatin (DDP). Figure 1 As shown in A, they were 20.09±1.06μM and 108.4±6.01μM, respectively. The IC50 of the two cells was significantly different, and the resistance coefficient was about 5.4. The qRT-PCR experiment detected the expression of AKT pathway-related genes AKT1, FOXO3, GSK3B and autophagy-related genes SQSTM1 and MAP1LC3B in the two cells at the mRNA level. Figure 1 As shown in B, compared with the parental cells, the expression of AKT1, FOXO3, GSK3B, SQSTM1 and MAP1LC3B genes in DDP-resistant cells was significantly increased. Western blotting was further used to detect the levels of proteins related to the AKT pathway and autophagy pathway, such as Figure 1 As shown in C, compared with MDA-MB-231 parental cells, the expression of AKT and p-AKT (Ser 473, Thr 308) in MDA-MB-231 / DDP resistant cells increased; the protein levels of GSK3β, p-GSK3β, and p-FOXo3a downstream of AKT also increased, and the level of FOXo3a decreased. Figure 1 As shown in D, the protein levels of autophagy-related proteins LC3II, ATG7, and Beclin1 increased in breast cancer DDP-resistant cells, and the autophagy substrate P62 decreased significantly. In the immunofluorescence experiment of p62, Figure 1 As shown in E, the average fluorescence intensity of p62 in MDA-MB-231 / DDP-resistant cells decreased significantly compared with that in MDA-MB-231 parental cells. The above results prove that compared with MDA-MB-231 parental cells, the AKT pathway and autophagy are activated in MDA-MB-231 / DDP-resistant cells.

[0110] Example 2

[0111] The inventors' preliminary experiments found that the AKT pathway and autophagy were activated in MDA-MB-231 / DDP-resistant cells. Figure 2As shown in A&2B, Western blotting indicated that under the action of cisplatin concentration gradient, the level of AKT pathway protein p-AKT (Ser473, Thr308) in MDA-MB-231 and MDA-MB-231 / DDP-resistant cells increased significantly, and the protein levels of AKT downstream GSK3β, p-GSK3β, FOXo3a, and p-FOXo3a also increased; as shown in Fig.2C&2D, the autophagy-related protein LC3II / LC3I increased, and the autophagy substrate P62 decreased. The above experimental results prove that cisplatin activates the AKT pathway and autophagy in MDA-MB-231 and MDA-MB-231 / DDP-resistant cells. DDP activates the AKT pathway and autophagy in MDA-MB-231 parental and DDP-resistant cells.

[0112] Example 3

[0113] like Figure 3 As shown in A, the MTT experiment proved that compared with the DDP monotherapy group, the proliferation activity of MDA-MB-231 / DDP cells in the DDP combined with Triciribine (AKT inhibitor) group was significantly reduced. The inventors further used cell cloning and EdU experiments to verify the sensitivity of MDA-MB-231 / DDP resistant cells to DDP after AKT pathway inhibition. Figure 3 As shown in B, the number of clones in the DDP and Triciribine combination group was significantly reduced; Figure 3 C shows that the proportion of green fluorescence in the combined group decreased, and the proportion of newborn cells was significantly reduced. Figure 3 DWestern blotting was used to detect the effect of DDP combined with Triciribine on AKT and apoptosis pathways. Compared with the DDP monotherapy group, the p-AKT (Ser 473, Thr 308) in the combined group was significantly reduced, and the apoptosis-related proteins cl-PARP and cl-Caspase3 were increased. The above experiments show that after the inhibition of the AKT pathway, the sensitivity of triple-negative breast cancer MDA-MB-231 / DDP cells to DDP is increased.

[0114] Example 4

[0115] Since the AKT inhibitor Triciribine can increase the sensitivity of DDP to MDA-MB-231 / DDP cells, the inventors further used siRNA of the AKT gene to verify the relationship between AKT and the anti-tumor effect of DDP. Figure 4 As shown in A, among the three knockdown sequences of AKT gene, #3 had the best knockdown effect, so this sequence was used for subsequent experiments. Cell cloning and EdU experiments revealed that Figure 4 As shown in B and 4C, the cell proliferation ability in the combined group was significantly decreased. Figure 4 As shown in Figure D, MTT experiments were further used to verify that compared with the DDP monotherapy group, the proliferation activity of MDA-MB-231 / DDP cells in the DDP and AKT siRNA combination group was significantly reduced. Western blotting experiments were used to detect the effects of DDP combined with AKT siRNA on the AKT pathway and cell apoptosis. Figure 4 As shown in E, compared with the DDP monotherapy group, the combined group AKT and p-AKT (Thr 308) were significantly reduced, and the apoptosis-related proteins cl-PARP and cl-Caspase3 were increased. Annexin V-FITC / PI double staining was used to further detect the effect of DDP combined with AKT siRNA on the apoptosis rate of MDA-MB-231 / DDP cells. Figure 4 As shown in F, compared with the 33.01% apoptosis rate in the DDP monotherapy group, the apoptosis rate in the combined group increased to 62.75%. The above experiments show that knocking down the AKT gene can increase the sensitivity of breast cancer DDP-resistant cells.

[0116] Example 5

[0117] Several studies have shown that blocking autophagy can increase the sensitivity of drug-resistant cells to chemotherapeutic drugs. Figure 5 As shown in A, the MTT experiment showed that compared with the DDP monotherapy group, the proliferation activity of MDA-MB-231 / DDP cells in the DDP combined with CQ (autophagy inhibitor) group was significantly reduced. Figure 5 As shown in B, the proportion of green fluorescence decreased in the combined group, and the proportion of newborn cells decreased significantly; Figure 5 C shows that the number of clones in the combined group of DDP and CQ was significantly reduced. Figure 5 DWestern blotting experiments were used to detect the effects of DDP combined with CQ on autophagy and apoptosis pathways. Compared with the DDP monotherapy group, the autophagy substrate p62 and LC3 in the combined group increased significantly, and the apoptosis-related proteins cl-PARP and cl-Caspase3 increased. The above experiments show that after blocking autophagy, the sensitivity of breast cancer DDP-resistant cells to DDP is increased.

[0118] Example 6

[0119] The inventors used different concentrations of EM2 to act on normal breast epithelial cells MCF-10A, normal liver epithelial cells LO2, breast cancer MDA-MB-231 parental cells and DDP-resistant cells for 48 hours, and the MTT method was used to detect the proliferation inhibition effect. Figure 6As shown in A, the 50% inhibition rate (IC50) of EM2 was 23.48±1.45, 34.63±3.97, 11.28±0.62, and 18.43±0.60 μM, respectively. Breast cancer MDA-MB-231 parental and DDP-resistant cells were treated with different concentrations of EM2 for 7 days. Figure 6 As shown in B and 6C, the results of the cell clone formation experiment indicate that as the concentration of EM2 increases, the inhibitory effect on cell clone formation becomes more significant. Figure 6 As shown in D, EdU experiments further verified the inhibitory effect of EM2 on breast cancer cell proliferation. Western blotting experiments detected apoptosis-related proteins, such as Figure 6 As shown in E&6F, EM2 was found to promote apoptosis of breast cancer MDA-MB-231 parental and DDP-resistant cells. Annexin-FITC / PI double staining flow cytometry was used to further verify the pro-apoptotic effect of EM2. Figure 6 As shown in G&6H, with the increase of EM2 concentration, the apoptotic proportion of breast cancer MDA-MB-231 parental and DDP-resistant cells increased significantly.

[0120] Example 7

[0121] Western blotting was used to detect the effect of EM2 on the AKT pathway in breast cancer MDA-MB-231 parental cells and DDP-resistant cells. Figure 7 As shown in A&7B, with the increase of EM2 concentration, the levels of AKT and p-AKT (Ser473, Thr308) decreased significantly, and the protein levels of GSK3β, p-GSK3β, FOXo3a, and p-FOXo3a, which are downstream of AKT, also decreased; Figure 7 As shown in &7D, the ratio of LC3II / LC3I decreased, and the autophagy substrate p62 increased significantly. qRT-PCR was further used to detect the expression of AKT pathway-related genes AKT1, FOXO3, GSK3B and autophagy-related genes SQSTM1, MAP1LC3B, and ATG5 at the mRNA level. Figure 7 As shown in E, in MDA-MB-231 / DDP-resistant cells, the expression of AKT1, FOXO3, and GSK3B genes was significantly reduced. At the same time, it was found that ATG5 was reduced, but SQSTM1 expression was increased, and MAP1LC3B had no significant change. Immunofluorescence was used for further verification, as shown in Figure 2. Figure 7 As shown in F, the average fluorescence intensity of p62 increased in a concentration-dependent manner and was distributed in an aggregated manner. The above experiments proved that EM2 is a dual inhibitor of AKT pathway and autophagy in breast cancer MDA-MB-231 parental and DDP-resistant cells.

[0122] Example 8

[0123] The inventors used different concentrations of DDP with or without low doses (IC20-IC30) of EM2 to act on MDA-MB-231 parental and DDP-resistant cells for 48 h. Figure 8 As shown in A&8C and Table 1, the MTT assay found that the IC50 of MDA-MB-231 cells to DDP decreased from 20.09±1.06μM to 5.73±0.29μM; the IC50 of MDA-MB-231 / DDP cells to DDP decreased from the original 108.4±6.01μM to 4.643±0.04μM, a 23.35-fold increase in sensitivity, completely reversing drug resistance. As shown in Fig.8B&8D, the combined group had a significant inhibitory effect on cell clone formation. Figure 8 As shown in E, the EdU experiment further proved that the proportion of newborn cells in the combined group was significantly reduced. Annexin-FITC / PI double staining flow cytometry was used for cell flow cytometry analysis to further verify that Figure 8 As shown in Figures F&8G, the apoptosis rate of breast cancer MDA-MB-231 parental and DDP-resistant cells in the combined group was significantly increased. The above experimental results prove that EM2 can increase the sensitivity of TNBC cells to DDP and reverse drug resistance. The IC50 and sensitization multiples of DDP and combined treatment with EM2 on MDA-MB-231 and MDA-MB-231 / DDP cells are shown in Table 5.

[0124] Table 5

[0125]

[0126]

[0127] Example 9

[0128] The inventors used DDP in combination with AKT inhibitor Triciribine or autophagy blocker CQ to increase the sensitivity of MDA-MB-231 / DDP-resistant cells to DDP by inhibiting the AKT pathway or blocking autophagy. EM2 is a dual inhibitor of the AKT pathway and autophagy, so we speculate that EM2 combined with DDP reverses the resistance of MDA-MB-231 / DDP-resistant cells to DDP by simultaneously blocking the AKT pathway and blocking autophagy. Fig. 9 As shown in A&9B, the expression level of p-AKT (Thr308, Ser473) in the DDP and EM2 combination group was significantly lower than that in the DDP monotherapy group, and the expression of p-GSK3β and p-FOXo3a, the downstream proteins of the AKT pathway, was also significantly reduced. Fig. 9 In C&9D, the expression of p62 increased in the DDP and EM2 combination group. Western blotting assay was used to detect apoptosis-related proteins, such as Fig. 9 As shown in E&9F, it was found that DDP combined with EM2 could promote apoptosis of breast cancer MDA-MB-231 parental and DDP-resistant cells, and the expression of cl-PARP increased in the combined group. The above experimental results prove that EM2 can inhibit the AKT pathway activated by DDP and protective autophagy, promote cell apoptosis, thereby increasing DDP sensitivity and reversing drug resistance.

[0129] In summary, compared with parental cells, AKT pathway activation and increased autophagy were observed in human triple-negative breast cancer MDA-MB-231 / DDP-resistant cells. Cisplatin can activate the AKT pathway in human triple-negative breast cancer cells and activate protective autophagy. Inhibiting the AKT pathway or blocking autophagy can increase the sensitivity of MDA-MB-231 / DDP-resistant cells to cisplatin. The monomer EM2 of Euphorbia pulegnifolia has a good anti-breast cancer effect and is less toxic to normal epithelial cells. EM2 can inhibit the AKT pathway in breast cancer cells and block cell autophagy. Low-dose EM2 combined with cisplatin can improve the anti-tumor effect of cisplatin and completely reverse drug resistance by blocking the AKT and autophagy pathways activated by cisplatin.

[0130] The above-described embodiments only express several embodiments of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

1. Use of a sesquiterpene lactone compound in the preparation of a drug for treating or adjuvanting triple-negative breast cancer, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

2. Use of a sesquiterpene lactone compound in the preparation of a drug for inhibiting the proliferation of triple-negative breast cancer cells, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

3. Use of a sesquiterpene lactone compound in the preparation of a compound for blocking cisplatin-activated AKT pathway and enhancing autophagy, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

4. Use of a sesquiterpene lactone compound in the preparation of an anti-triple-negative breast cancer synergist, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

5. Use of a sesquiterpene lactone compound in the preparation of an agent for reversing triple-negative breast cancer resistance, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

6. Use of a sesquiterpene lactone compound in combination with cisplatin in the preparation of a drug for the treatment or adjuvant treatment of triple-negative breast cancer, wherein the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof:

7. A pharmaceutical composition for treating triple-negative breast cancer, characterized in that: include: The sesquiterpene lactone compound represented by formula (I), its pharmaceutically acceptable salt, its stereoisomer or its prodrug molecule:

8. A pharmaceutical composition according to claim 7, characterized in that The dosage form is selected from: Injection, tablet, capsule, kit or patch.

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