Application of sesquiterpene lactone compound in preparation of medicine for treating p53 wild type ovarian cancer

Abstract

The invention discloses application of a sesquiterpene lactone compound in preparation of a medicine for treating p53 wild type ovarian cancer, an elephantopus tomentosus monomer EM-12 shown in a formula (I) is a novel sesquiterpene lactone compound extracted and identified from elephantopus tomentosus, has a good proliferation inhibition effect on p53 wild type ovarian cancer cells, and can be used for preparing a medicine for treating p53 wild type ovarian cancer cells. The toxicity to normal ovarian epithelium IOSE80 cells is relatively low; on the other hand, the EM-12 can promote dissociation of MDM2 and p53 and inhibit a proteasome degradation pathway of p53 by targeting MDM2, and meanwhile, the EM-12 blocks autophagy flux and inhibits a lysosome degradation pathway of p53; the p53 level in the cell is increased, and accumulation of p53 induces G1 / S phase retardation of the A2780 cell, so that cell apoptosis is caused, and the anti-tumor effect is achieved.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and specifically to the application of sesquiterpene lactone compounds in the preparation of drugs for treating p53 wild-type ovarian cancer. Background Art

[0002] Ovarian cancer is one of the most common gynecological malignancies in women, characterized by high incidence and high mortality. As an important tumor suppressor gene, the p53 gene plays a key role in the occurrence and development of ovarian cancer. Patients with p53 wild-type ovarian cancer account for about 50% of all ovarian cancer patients, and the expression level of p53 protein in their tumor cells is closely related to the prognosis of the patients. However, there are still many limitations in the current treatment methods for p53 wild-type ovarian cancer, such as the large toxic and side effects of chemotherapy drugs and the easy generation of drug resistance in tumor cells, which seriously restrict the treatment effect.

[0003] Despite the rapid progress in platinum-based chemotherapy, drugs targeting poly(ADP-ribose) polymerase (PARP), and immunotherapy, the clinical efficacy of ovarian cancer patients is still not ideal because most ovarian cancer patients are diagnosed with locally advanced and widely metastatic tumors (stage III and stage IV). In addition, the emergence of drug-resistant cells will lead to recurrence after the end of treatment, and the prognosis of ovarian cancer patients with recurrence and drug resistance is often worse. Therefore, the research of new anti-ovarian cancer drugs is of great significance for the clinical treatment of ovarian cancer and improving the survival rate of patients. Summary of the Invention

[0004] This application aims to provide the application of sesquiterpene lactone compounds in the preparation of drugs for treating p53 wild-type ovarian cancer.

[0005] The technical solution adopted by the present invention is as follows:

[0006] In the first aspect of this application, there is provided the application of sesquiterpene lactone compounds in the preparation of drugs for treating or adjuvantly treating p53 wild-type ovarian cancer, wherein the sesquiterpene lactone compounds are the compounds shown in formula (I), their pharmaceutically acceptable salts, their stereoisomers or their prodrug molecules:

[0007]

[0008] More specifically, in the above technical solution, the drug includes the following characteristics: targeting MDM2 and inhibiting the proteasomal degradation of p53.

[0009] More specifically, in the above technical solution, the drug further includes the following characteristics: arresting the cancer cells of p53 wild-type ovarian cancer in the G1 / S phase of the cell cycle.

[0010] More specifically, in the above technical solution, the dosage form of the drug is selected from: injection, tablet, capsule, kit or patch.

[0011] In a second aspect, the present application provides an application of a sesquiterpene lactone compound in the preparation of a drug for inhibiting the proliferation of wild-type p53 ovarian cancer cells. The sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

[0012]

[0013] More specifically, in the above technical solution, the wild-type p53 ovarian cancer cells include: A2780, OVCAR-5, OVCAR-8, TOV-112D, OVCAR-3 and SK-OV-3.

[0014] More specifically, in the above technical solution, preferably, the wild-type p53 ovarian cancer cells are: A2780, OVCAR-5 and OVCAR-8.

[0015] In a third aspect, the present application provides an application of a sesquiterpene lactone compound in the preparation of an apoptosis blocker for inducing wild-type p53 ovarian cancer cells. The sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

[0016]

[0017] In a fourth aspect, the present application provides an application of a sesquiterpene lactone compound in the preparation of a synergist for anti-wild-type p53 ovarian cancer. The sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug molecule thereof:

[0018]

[0019] The "pharmaceutically acceptable salts" are conventional non-toxic salts formed by the reaction of the compound of general formula (I) with inorganic acids or organic acids. For example, the conventional non-toxic salts can be prepared by reacting the compound of general formula (I) with inorganic acids or organic acids. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid, etc., and the organic acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic 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, hydroxyethanesulfonic acid, etc.; or the sodium salts, potassium salts, calcium salts, aluminum salts or ammonium salts formed by reacting the compound of general formula (I) with inorganic bases after forming esters 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; or the methylamine salts, ethylamine salts or ethanolamine salts formed by reacting the compound of general formula (I) with organic bases; or the corresponding inorganic acid salts formed by reacting the compound of general formula (I) with lysine, arginine, ornithine to form esters and then reacting with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid or the corresponding organic acid salts formed by reacting with formic acid, acetic acid, picric acid, methanesulfonic acid and ethanesulfonic acid.

[0020] 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 drug compositions into various dosage forms according to specific circumstances, and the preparation method is well-known in the art, so it will not be elaborated in this application.

[0021] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0022] 1. The inventors first found through MTT experiments that EM-12 has relatively low toxicity to the normal ovarian epithelial cell line IOSE80, and EM-12 significantly inhibits the cell viability of the p53 wild-type ovarian cancer cell line. The half-inhibitory concentration IC 50 (72h) of EM-12 against three p53 wild-type ovarian cancer cells A2780, OVCAR-5, and OVCAR-8 are 1.41±0.15 μM, 2.885±0.19 μM, and 4.64±1.96 μM, respectively.

[0023] 2. EM-12 can target MDM2, promote the dissociation of MDM2 from p53, inhibit the proteasome degradation pathway of p53, resulting in an increase in the intracellular p53 level. The accumulation of p53 induces G1 / S phase arrest in A2780 cells, causing apoptosis, thereby exerting an anti-tumor effect.

[0024] 3. EM-12 can block autophagic flux, inhibit the lysosomal degradation pathway of p53, leading to an increase in intracellular p53 levels. The accumulation of p53 induces G1 / S phase arrest in A2780 cells, causing apoptosis, thereby exerting an anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0026] Figure 1 It is the high-resolution electrospray mass spectrum of EM-12.

[0027] Figure 2 EM-12 has different inhibitory effects on the proliferation of breast cancer cells under different p53 states. A. Chemical structure of EM-12. B. Human normal ovarian epithelial cells IOSE80 and several human ovarian cancer cell lines were treated with different concentrations of EM-12 for 72 h, and then the cell viability was detected by the MTT method. C. IC50 (72 h) of EM-12 for different cell lines.

[0028] Figure 3 It is the action target of EM-12 in ovarian cancer. A. The binding protein of EM-12 was found by the Drug Affinity Responsive Target Stability technology, and three pronase concentrations were used in the experiment. High concentration of pronase reduced the protein content with a molecular weight of 90 kDa. B. The molecular docking model was used to simulate the effect of EM-12 on the interaction between MDM2 and p53. C. Western blotting was used to detect the protective effect of EM-12 on MDM2 protein in the presence of pronase.

[0029] Figure 4 The expression of p53 affects the anti-tumor effect of EM-12 on ovarian cancer cells. A. The cell survival rate after EM-12 (0 or 2 μmol / L) treatment for 72 h was detected by the MTT method in three p53 wild-type ovarian cancer cells A2780, OVCAR-5, and OVCAR-8 transfected with the control group and p53 siRNA, respectively. B. In the p53-deficient cell line SKOV3 (p53- / -), Flag-p53-WT or Flag-p53-MT plasmids were transfected respectively, and after EM-12 (0 or 2 μmol / L) treatment for 72 h, the cell viability was detected by the MTT method.

[0030] Figure 5EM-12 promotes the dissociation of MDM2 from p53. A. After A2780 cells were treated with EM-12 for 24 h, immunoprecipitation was performed using anti-p53 antibody and Protein A / G Plus-Agarose Beads, and the expression level of anti-MDM2 was detected by Western blotting. B. 293T cells were transfected with pcDNA3.1B-HA-Ub and Flag-p53 plasmids. After treatment with EM-12 and the proteasome inhibitor MG132, the ubiquitination level of P53 was observed by immunoprecipitation. C. The effect of EM-12 on its half-life in the presence of CHX. D. The MDM2 protein levels in A2780, OVCAR-5, OVCAR-8, and LO2 cells were detected by Western blotting.

[0031] Figure 6 EM-12 promotes the nuclear translocation of p53 protein. A. After immunofluorescence staining, the distribution of P53 in the cytoplasm and nucleus was observed by laser confocal microscopy. B. After treatment with MG132, EM-12 was applied to A2780 cells for 24 h, and then the distribution of p53 in the cytoplasm and nucleus was detected by Western blotting.

[0032] Figure 7 EM-12 inhibits the autophagic degradation pathway of p53 by blocking autophagy. A and B. After treatment with 0, 1, 2, 3 μM EM-12 for 24 h, the expression levels of autophagy-related proteins in cells were detected by Western blot. C. After treatment with 2 μM EM-12 for 0, 3, 6, 12, 24, 36 h in A2780 cells, the expression levels of autophagy-related proteins in cells were detected by Western blot. D. 3 μM EM-12 was applied to A2780 cells transfected with GFP-mCherry-LC3 for 24 h. Scale bar: 10 μm. E. Autophagy was labeled with LC3 and lysosomes were labeled with LAMP2, and then after treatment with 3 μM EM-12 for 24 h, observations were made under a confocal microscope. F. A2780 cells were transfected with siAtg5, and the expression levels of p53 under different serum conditions were detected by Western blotting.

[0033] Figure 8EM-12 induces cell cycle arrest and apoptosis in A2780 cells. A. After A2780 cells were treated with EM-12 for 24 h, the cells were stained with PI and detected by flow cytometry. B and C. After the cells were treated with 0, 1, 2, 3 μM EM-12 for 24 h, the expression levels of cell cycle-related proteins were analyzed by Western blot (B). The mRNA levels of p21, Noxa and p53 were analyzed by qRT-PCR (C). D. After the cells were treated with 0, 1, 2, 3 μM EM-12 for 24 h, the expression levels of apoptosis-related proteins were detected by Western blot.

[0034] Figure 9 EM-12 induces apoptosis in A2780 cells. A. After A2780 cells were treated with different concentrations of EM-12 for 24 h, the cells were stained with FITC-cl-Caspase-3 and analyzed by flow cytometry. B. The EdU method was used to detect the proliferation inhibitory effect of EM-12 on A2780 cells. A2780 cells were treated with different concentrations of EM-12 for 24 h, and the cells treated with DMSO at a volume ratio of 0.02% were used as the control group, scale bar: 250 μm. C. The Tunel method was used to detect apoptosis in A2780 cells treated with different concentrations of EM-12 for 24 h, scale bar: 100 μm.

[0035] Figure 10 EM-12 induces the accumulation and apoptosis of p53 in cancer cells. A and B. After the cells were treated with 0, 1, 2, 3 μM EM-12 for 24 h, the expression levels of p53 and PARP were detected by Western blot. Detailed implementation mode

[0036] In order to more clearly understand the technical content of the present invention, the following specific examples are given in detail in conjunction with the accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0037] The monomer EM-12 is a novel sesquiterpene lactone compound extracted from the traditional Chinese herbal medicine Elephantopus scaber L., and its chemical formula is C 20 H 26 O7, and the molecular weight is 378.42. The chemical structural formula of EM-12 is shown in formula (I). Figure 1 The high-resolution mass spectrum of EM-12 is as follows: (HRESIMS: m / z measured molecular weight 401.1571 [M + Na]+ (theoretical value: 401.1571), showing the molecular formula C 20 H26 O7. The EM-12 stock solution was prepared with DMSO at a concentration of 100 mM, aliquoted and stored at -80 °C.

[0038]

[0039] 1. Experimental Materials and Methods

[0040] Cell lines: Human ovarian cancer cells A2780 (p53wt), OVCAR-5 (p53wt), OVCAR-8 (p53wt), TOV-112D (p53R175H), OVCAR-3 (p53R248W), SK-OV-3 (p53- / -), human normal ovarian epithelial cells IOSE80 and human normal liver cells LO2 were all purchased from the American Type Culture Collection (ATCC).

[0041] Methods: MTT assay was used to detect the proliferation inhibitory effect of EM-12 on cancer cells with different p53 expression statuses and its cytotoxic effect on human normal cells. Enzyme hydrolysis experiment, immunoblotting and molecular docking model were used to verify the action targets of EM-12; blocking experiment and rescue experiment were used to prove that EM-12 exerts anti-tumor effects through p53. Immunofluorescence, immunoblotting and immunoprecipitation were used to detect the effect of EM-12 on the interaction between MDM2 and p53. Immunoblotting, immunofluorescence, qRT-PCR and autophagy double-labeling system (mCherry-GFP-LC3) were used to detect the effect of EM-12 on autophagy. Flow cytometry and immunoblotting were used to detect the effect of EM-12 on cell cycle and apoptosis.

[0042] MTT Assay:

[0043] (1) Cells in the logarithmic growth phase were digested with trypsin. After about 1 - 3 minutes, the digestion was terminated by adding the culture medium containing 10% FBS. The cells were centrifuged at 800 rpm for 3 minutes at room temperature, and then the supernatant was removed. The cells were resuspended with the culture medium containing 10% FBS to make a single-cell suspension and counted. A 96-well plate was taken, and 100 μL of the cell suspension was added at a density of 5000 cells per well. Each group was set with 3 - 5 replicates, and the outer circle of the well plate was filled with 200 μL of 1×PBS to reduce the evaporation of the culture medium, and then placed in the incubator for culture.

[0044] (2) After overnight adherence, the drug was prepared by the equal ratio dilution method. 100 μL of the culture medium containing the drug was added to each well to make the total volume in the well 200 μL. The 96-well plate was gently shaken to make the drug evenly distributed, and then cultured in the incubator for 48 hours.

[0045] (3) After 48 hours, add 20 μL of MTT solution to each well and continue to incubate it in an incubator at 37 °C for 4 - 6 h.

[0046] (4) After 4 - 6 h, aspirate the mixture of culture medium and MTT completely with a vacuum suction pump, and add 150 μL of DMSO solution to each well. After the formazan purple crystals are fully dissolved, use a multifunctional microplate reader to detect the absorbance at 490 nm. Calculate the survival rate of the cells in each group by calculating the average absorbance value of each group. Calculate the IC50 (Mean ± SD) using IBM SPSS Statistics 20 and plot the graph using GraphPad Prism 7.0.

[0047] Cell colony formation assay:

[0048] (1) Take cells in the logarithmic growth phase, digest the cells with trypsin, centrifuge at low speed, discard the supernatant, and resuspend with culture medium. After cell counting, seed the cells evenly into a six-well plate at about 500 - 1000 cells per well. Gently shake the six-well plate and observe under the microscope to ensure that the cells are evenly seeded in the six-well plate, and then place it in a cell culture incubator.

[0049] (2) After the cells adhere to the wall, remove the culture medium, and add 2 mL of culture medium containing different concentrations of drugs to each well to treat the cells, and continue to culture for 7 - 14 days.

[0050] (3) After the cell colony diameter reaches 0.3 mm to 1 mm, remove the culture medium, wash 2 - 3 times with PBS, add an appropriate amount of paraformaldehyde solution to each well to completely cover the cells, fix at room temperature for 20 min, then remove the fixing solution and wash with PBS. Dilute the 1% crystal violet staining solution to 0.1% with PBS, add 1 mL to each well for staining for 30 min, remove the staining solution, then gently rinse the well plate with running water, dry it, and take pictures or scan to save the pictures.

[0051] qRT-PCR assay:

[0052] (1) Total RNA extraction - TRIzol method: To prevent RNA enzyme contamination, wear masks, gloves, and headgear throughout the experiment, and ensure that all consumables and reagents used in the experiment are free of RNA enzyme contamination.

[0053] After the drug acts on the cells, aspirate the supernatant of the culture medium and wash twice with pre-cooled 1×PBS.

[0054] In a biosafety cabinet, add 1 mL of TRIzol to each well of the six-well plate to lyse the cells. Let it stand for 5 min, transfer it to a 1.5 mL enzyme-free centrifuge tube, and then pre-cool the high-speed refrigerated centrifuge in advance.

[0055] Add 200 μL of chloroform according to the ratio of TRIzol:chloroform = 5:1. After vigorously oscillating for 15 s with a vortex mixer, let it stand still for another 3 min. After observing the liquid phase separation, use a high-speed refrigerated centrifuge to centrifuge at 4°C at a speed not exceeding 12,000 rpm for 15 min.

[0056] After centrifugation, transfer the upper aqueous phase to a new nuclease-free microcentrifuge tube. Add 500 μL of isopropanol according to the ratio of TRIzol:isopropanol = 2:1, and continue to centrifuge at 4°C at a speed not exceeding 12,000 rpm for 10 min.

[0057] After observing a white precipitate at the bottom of the tube with the naked eye, discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water to wash the precipitate, and continue to centrifuge at 4°C at a speed not exceeding 7,500 rpm for 5 min. Repeat the washing twice.

[0058] Aspirate all the 75% ethanol, open the lid of the centrifuge tube, and invert it in a 37°C drying oven to completely volatilize the 75% ethanol. When observing that the white precipitate is about to become transparent, add nuclease-free water to dissolve the RNA precipitate. Use a NanoDrop 2000 micro ultraviolet spectrophotometer to detect the concentration and purity of RNA.

[0059] (2) Reverse transcription-PCR (RT-PCR): A total of 20 μL system. After mixing, incubate at 37°C for 15 min and at 85°C for 5 s, and store the samples at -20°C.

[0060] (3) Real-time fluorescence quantitative PCR:

[0061] 1) Premixed solution system: 20 μL. Set 3 replicates for each gene detected in each treatment sample. The cDNA template comes from the product of the reverse transcription system and is diluted tenfold for use.

[0062] 2) qPCR program: Three-step method

[0063] Pre-denaturation: 95°C for 10 min; Amplification (40 cycles): Denaturation: 95°C for 15 s, Annealing: 60°C for 30 s, Extension: 72°C for 30 s; The melting curve program uses the default settings of the instrument.

[0064] Flow cytometry experiment:

[0065] (1) Take out the 6-well plate after treatment with the corresponding drug, and collect the culture medium in the wells into a centrifuge tube. Wash each well twice with 1 mL of 1×PBS, and collect the aspirated PBS into the centrifuge tube as well. Note that the operation should be gentle to avoid physical damage to the cells.

[0066] (2) Add an appropriate amount of trypsin without EDTA to each well to digest the cells. Transfer the cells to a centrifuge tube and centrifuge at 800 rpm for 3 min.

[0067] (3) Discard the supernatant, add 1 mL of PBS to resuspend the cells. Note that do not pipette forcefully, and must be gentle to avoid damaging the cells. Centrifuge at 800 rpm for 3 min. Repeat 2 times until the residual culture medium is washed away, and then transfer to a 1.5 mL centrifuge tube.

[0068] (4) Discard the supernatant, add 300 μL of Binding Buffer to each tube to resuspend the cells. Add 3 μL of AnnexinV staining solution and mix well. Incubate at room temperature for 10 minutes, then add 3 μL of PI staining solution and mix well. Incubate at room temperature in the dark for 10 minutes, and then perform the detection on the machine as soon as possible.

[0069] SiRNA experiment:

[0070] (1) Seed cells in the logarithmic growth phase into a six-well plate, culture with medium without antibiotics, and incubate overnight until the cells adhere to the wall.

[0071] (2) Dilute 4 μL of siRNA and 4 μL of LipfectaminTM 2000 separately in 250 μL of serum-free basal medium, and place at room temperature for 5 min.

[0072] (3) Gently mix the diluted siRNA solution with the liposome solution, and place at room temperature for 20 min.

[0073] (4) Aspirate the complete medium without antibiotics from the cell culture, and rinse the cells 2 times with PBS.

[0074] (5) Add the prepared siRNA-liposome complex to the cells, gently shake the six-well plate back and forth to disperse the mixture evenly, and then add 500 μL of serum-free basal medium Opti-MEM.

[0075] (6) After 4 - 6 h, aspirate the transfection complex and replace it with fresh medium containing 10% fetal bovine serum.

[0076] (7) Continue to culture in a CO2 incubator for 24 - 48 h, and then perform subsequent experiments.

[0077] Plasmid transfection experiment:

[0078] (1) Plasmid transformation

[0079] Take out DH5α competent cells (50 μL) from the -80 °C refrigerator, place them on ice to thaw, and preheat the water bath to 42 °C in advance.

[0080] Add the plasmid to be transformed into the competent cells, gently flick the centrifuge tube to mix it evenly, avoid pipetting up and down, and incubate on ice for 30 min.

[0081] Heat shock in a water bath at 42 °C for 40 s, quickly place the EP tube on ice to cool for 2 min to bring the temperature down to 0 °C.

[0082] Add 800 μl of LB medium without antibiotics to the EP tube, gently shake on a shaker at 37 °C for 1 h (150 rpm). When shaking, note that the inside of the tube should be in contact with air to allow the competent cells to resume normal growth and express the ampicillin resistance gene for screening.

[0083] Take 150 μl of the bacterial solution in a sterile operating bench and evenly inoculate it on the LA solid medium containing ampicillin. Place it at room temperature for 2 - 3 min. After the liquid is fully absorbed, make a mark and invert it in an incubator at 37 °C for about 16 hours.

[0084] (2) Plasmid extraction

[0085] Take out the LA solid medium that has been cultured for 16 h above, pick a single colony in a sterile operating bench and put it into 5 ml of LB liquid culture medium containing ampicillin (100 μl / ml). When operating, light an alcohol lamp to prevent contamination by miscellaneous bacteria, and shake and culture overnight in an incubator at 37 °C.

[0086] The next day, take out 3 ml of the bacterial solution (the remaining part is used for medium-scale plasmid extraction), centrifuge at 12,000 rpm for 3 min, and discard the supernatant.

[0087] Add 300 μl of Buffer P1 to resuspend the pellet; then add 300 μl of Buffer P2. At this time, the liquid turns blue. Invert the EP tube to mix evenly, avoid shaking, incubate at room temperature for 5 minutes, and then add 300 μl of Buffer P3. It can be seen that the blue liquid becomes lighter and layers. After inverting and mixing, the liquid becomes white flocculent, and incubate on ice for 5 minutes.

[0088] Centrifuge at 16,000 rpm for 15 min; take out the supernatant and add it to the QIAprep spin column, let it stand for 2 min; centrifuge at 13,000 rpm for 1 min, and discard the filtrate.

[0089] Add 0.75 ml of Buffer PE to wash the QIAprep spin column, let it stand for 2 min, centrifuge at 13,000 rpm for 1 min, and discard the filtrate.

[0090] Centrifuge the empty QIAprep spin column at 13,000 rpm for 1 min.

[0091] Place the QIAprep spin column in a clean 1.5 ml EP tube, then add 50 μl of ddH2O and let it stand for 2 - 3 min. Finally, centrifuge at 13,000 rpm for 1 min to collect the eluate.

[0092] Use a nucleic acid and protein analyzer (Eppendorf) to measure the DNA concentration and purity of the extracted plasmid. Use 1% agarose gel electrophoresis to identify whether the extracted plasmid is correct.

[0093] (3) Plasmid transfection

[0094] Inoculate cells one day in advance to make the cell density 60% - 80% at the time of transfection. Replace the cells with fresh complete culture medium without double antibiotics before transfection.

[0095] Thaw the plasmid solution on ice in advance. Take the culture medium without double antibiotics and add it to two EP tubes respectively. Each system consists of two liquids of equal volume: one contains the plasmid and the other contains lipo3000; then mix the two tubes in equal volume and gently pipette to mix evenly to avoid damaging the plasmid structure.

[0096] After mixing the plasmid and the transfection reagent, let it stand at room temperature for 10 - 15 min. Label the well plate, and then add the corresponding plasmid dropwise to the cells. Gently shake the well plate to make the transfection system evenly distributed, and then put it into the incubator for further culture.

[0097] Western blot:

[0098] (1) Protein extraction and quantification

[0099] Inoculate 3.0×105 cells in good growth state into a 6-well plate. After overnight adherent culture, add drugs at corresponding concentrations and treat for 24 hours.

[0100] After the drug treatment, aspirate the culture medium, add pre-cooled 1×PBS for rinsing, and then add 100 - 200 μL of RIPA lysis buffer on ice according to the number of cells. The RIPA lysis buffer is pre-added with the protease inhibitor PMSF and the protein phosphatase inhibitor mixture. After lysing on ice for half an hour, collect the cell lysate into a non-enzymatic microcentrifuge tube. Pre-open the high-speed refrigerated centrifuge and separate the protein supernatant by centrifugation at 4℃, 12,000 rpm for 15 min. Transfer the protein supernatant to a new microcentrifuge tube and measure the protein concentration using a BCA kit.

[0101] After protein quantification, add SDS-PAGE protein loading buffer (6×) to dilute it to 1× and heat it in a 95℃ metal bath for 5 min. Aliquot the protein supernatant and store it at -80℃.

[0102] (2) Electrophoresis

[0103] According to the molecular weight of the target protein to be detected, prepare a polyacrylamide gel with an appropriate concentration. After assembling the electrophoresis apparatus, add 1× electrophoresis working solution. Then load the Rainbow Protein Marker and the sample protein onto the gel for electrophoresis. After electrophoresis at 80 V for 30 min, switch to 120 V and continue electrophoresis until the indicator band is close to the bottom of the gel.

[0104] (3) Transfer membrane

[0105] After electrophoresis, cut off the excess gel part and lay it flat on the transfer filter paper. The 0.22 μM PVDF membrane needs to be soaked and activated with methanol before use. After fitting it with the gel, remove the air bubbles, then insert it into the transfer clip with the gel side facing the black side of the transfer clip. Then, the black side of the transfer clip faces the negative electrode of the transfer tank, and the white side faces the positive electrode of the transfer tank. After filling the transfer tank with the electrotransfer solution, transfer the membrane at a constant current of 200 mA for 90 min, and fill the outer periphery of the transfer tank with ice cubes.

[0106] (4) Blocking

[0107] After the transfer is completed, soak the membrane in 5% blocking milk, gently shake it, and incubate it at room temperature for 1 h.

[0108] (5) Antibody incubation and chemiluminescence imaging

[0109] After blocking, wash off the excess milk with 1× TBST buffer. Dilute the primary antibody 1:1000 with the primary antibody diluent, then soak the membrane in the primary antibody and incubate it overnight at 4°C. After the primary antibody incubation, recover the primary antibody, add 1× TBST washing solution, place it on a shaker to wash the membrane, 10 min each time, for a total of 3 times. After washing the membrane, then place the PVDF membrane into the corresponding secondary antibody, incubate it at room temperature on a shaker for 1 - 2 h, then add TBST to wash the membrane, 10 min each time, for a total of 3 times. After washing the membrane, use the extra-sensitive ECL chemiluminescence solution to coat the membrane, expose it through a chemiluminescence imager, and collect and analyze the experimental results.

[0110] Coomassie Brilliant Blue staining experiment:

[0111] (1) Put the gel with Mark after electrophoresis into an appropriate amount of Coomassie Brilliant Blue staining solution for staining, ensuring that the staining solution can fully cover the gel.

[0112] (2) Place the gel on a horizontal shaker and gently shake it. Stain it at room temperature for at least 2 h. For low-abundance proteins, staining requires more than 4 h, and the staining time also needs to be extended at lower temperatures.

[0113] (3) When the gel can hardly be seen in the staining solution, it can be considered that the staining is sufficient. After staining, pour out the staining solution. The staining solution can usually be reused at least 2 - 3 times, but the staining effect will be slightly affected.

[0114] (4) Pour in an appropriate amount of decolorizing solution to ensure that the decolorizing solution can fully cover the gel. Place it on a shaker and shake slowly for 4 - 24 hours. During this period, change the decolorizing solution 2 - 4 times until the blue background is basically removed. Co-Immunoprecipitation experiment:

[0115] (1) Take cells in the growth phase, aspirate the culture medium, and wash the cells twice with PBS.

[0116] (2) Add 1 ml of pre-cooled RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, incubate at 4°C for 30 min, and tap the culture dish wall from time to time.

[0117] (3) Use a cell scraper to scrape the cells off the culture dish, and then transfer the lysate to a 1.5 ml EP tube.

[0118] (4) Centrifuge at 12,000 g for 15 min at 4°C, transfer the supernatant to a new centrifuge tube, being careful not to aspirate the white precipitate, and place it on ice.

[0119] (5) Perform protein quantification by the BCA method.

[0120] (6) According to the results of the BCA assay, take about 0.5 ml of the supernatant into a new 1.5 ml EP tube. It is necessary to ensure that the total protein amount in each tube is equal. Add an appropriate amount of primary antibody as needed, and mix well at 4°C for 1 - 2 h. At the same time, retain a small amount of the supernatant for Western blot analysis of the total protein.

[0121] (7) Take 50 μl of Protein A Sepharose 4 Fast Flow / Protein G Sepharose 4 Fast Flow (cut off the tip of the pipette to avoid damaging the agarose beads during operations involving agarose beads) into a DNase / RNase-free EP tube, add 1 ml of pre-cooled PBS for washing, centrifuge at 12,000 g for 30 s at 4°C, then let it stand for 1 min, discard the supernatant, and repeat 2 times.

[0122] (8) Add the protein suspension incubated with the primary antibody to the EP tube containing Protein A Sepharose 4 Fast Flow / Protein G Sepharose 4 Fast Flow, and mix well at 4°C for 1 h to overnight using a silent mixer.

[0123] (9) The next day, take out the centrifuge tube, centrifuge at 12,000 g for 30 s at 4°C, and let it stand for 1 min. To reduce the background and remove unbound proteins, try to aspirate the supernatant as much as possible, but it is better to leave a small amount of the supernatant rather than aspirate Protein A Sepharose 4 Fast Flow / Protein G Sepharose 4 Fast Flow.

[0124] (10) Wash the precipitate with PBS 3 - 5 times under the same centrifugation conditions as above.

[0125] (11) After the last wash, completely remove the supernatant. At this time, only the beads remain in the tube and there is no liquid. Add 45 μL of 1× SDS - PAGE loading buffer to each tube and mix well.

[0126] (12) Treat the sample at 95 - 100 °C for 10 min, shake the EP tube wall in the middle to fully mix the sample, and centrifuge at 12,000 g for 30 s after boiling the protein.

[0127] (13) Pipette 40 μL of the supernatant into a new EP tube, and take 20 μL of the supernatant to continue the experiment according to the corresponding Western Blot steps.

[0128] Immunofluorescence (IF) experiment:

[0129] (1) After the cells are treated with the corresponding drug, discard the culture medium, wash twice with PBS, add 200 μL of 4% paraformaldehyde, and fix the cells for 10 min.

[0130] (2) Aspirate the 4% paraformaldehyde, wash twice with PBS, then add 0.1% Triton - X and react at room temperature for 10 min.

[0131] (3) Add 10% goat serum to cover the cells and incubate at 37 °C for 1 h for blocking.

[0132] (4) Aspirate the 10% goat serum, wash with PBS, dilute the primary antibody at a ratio of 1:200 with 5% BSA to make a working solution, and incubate overnight at 4 °C. The primary antibody can be recycled and reused multiple times after incubation. Then wash the cells twice with PBS containing 2% Tween 20.

[0133] (5) Secondary antibody incubation: Add the corresponding fluorescent secondary antibody and incubate at room temperature for 1 h. The fluorescent secondary antibody is also diluted with 5% BSA at a dilution ratio of 1:100. After the incubation, aspirate the secondary antibody and wash with PBS containing 2% Tween 20 to remove the residual secondary antibody.

[0134] (6) Add 100 μL of ready - to - use DAPI staining solution, incubate at room temperature in the dark for 10 min, then wash twice with PBS, add an anti - fluorescence quenching agent to mount the slides to prevent fluorescence quenching. Observe the fluorescence under a laser scanning confocal microscope or store in the dark at 4 °C.

[0135] Detection of autophagic flux using the mCherry - GFP - LC3 autophagy dual - labeling system:

[0136] (1) Seed 5.0×10⁵ cells in good growth state in a 6-cm cell culture dish, add the culture medium, shake well, and let the cells adhere overnight.

[0137] (2) After the cells adhere, add 50 μL of lentivirus particles and 4 μg / μL of the infection-promoting reagent polybrene to infect the cells for 24 h. Then replace the fresh culture medium and add puromycin (5 μg / μL), and perform puromycin drug screening for 4 days, replacing the fresh culture medium containing 5 μg / μL of puromycin every day to kill the cells with low infection efficiency. After drug screening, observe and analyze the virus infection efficiency under a fluorescence microscope. When the infection efficiency is too low, increase the puromycin concentration to continue drug killing. When the efficiency reaches over 90%, it can be used for subsequent experiments.

[0138] (3) Seed the virus-infected cells into a glass-bottom culture dish. After they adhere, add EM-12 for treatment.

[0139] (4) After the drug treatment is completed, aspirate the culture medium containing the drug, add Hoechst 33342 to stain the cell nuclei for 15 min. After washing the residual Hoechst 33342 with pre-cooled PBS, replace it with fresh culture medium to cover the cells, and image and analyze the ratio of autophagosomes (Autophagosome, yellow) and autolysosomes (Autolysosome, red) under a laser scanning confocal microscope.

[0140] Cell proliferation detection - EdU method:

[0141] (1) Cell culture: Take cells in the logarithmic growth phase, add 100 μL of cell suspension to a 96-well plate at a density of 6000 cells per well, and set 3 replicates for each group.

[0142] (2) Drug treatment: After the cells adhere overnight, aspirate the culture medium, and then add the culture medium containing the corresponding concentration of the drug to each well and continue culturing for 24 h.

[0143] (3) EdU labeling of cells: After aspirating the original culture medium, add 100 μL of fresh culture medium containing 10 μM EdU working solution and continue incubating for 2 - 2.5 h.

[0144] (4) Cell fixation: After aspirating the EdU labeling solution, add 100 μL of 4% paraformaldehyde to each well to fix for 20 min. Remove the fixing solution, add 100 μL of glycine solution and incubate at room temperature for 5 min to remove the residual 4% paraformaldehyde.

[0145] (5) Cell permeabilization: Aspirate the glycine solution, add 100 μL of 3% BSA solution to wash, and then add 100 μL of 0.5% Triton X-100 and incubate at room temperature for 15 min.

[0146] (6) EdU assay: Aspirate 0.5% Triton X-100 and wash the cells with 3% BSA. Then add 100 μL of Click-iT reaction mixture prepared according to the reagent instructions and incubate in the dark at room temperature for 30 min.

[0147] (7) DNA counterstaining: Aspirate the reaction mixture, wash each well once with 100 μL of PBS, add 100 μL of 5 μg / mL Hochest 33342 solution, and incubate in the dark at room temperature for 15 min. Then wash with 100 μL of PBS and the cells can be monitored on the machine.

[0148] (8) Imaging and analysis: Take pictures and analyze under an inverted fluorescence microscope. kFluor488: Ex / Em = 495 nm / 519 nm, green fluorescence. Hoechst 33342 (bound to DNA): Ex / Em = 350 nm / 461 nm, blue fluorescence.

[0149] Detection of cell apoptosis by Tunel method:

[0150] (1) Select cells in good condition and seed them in plates, and culture them in a 37 °C incubator. The next day, remove the culture medium, add the corresponding drugs and treat for an appropriate time, make marks and continue to culture in a 37 °C constant temperature incubator for 72 hours.

[0151] (2) Perform experimental operations according to the cell apoptosis detection kit of KeyGen Biotech (product number: KGA7072).

[0152] Statistics and analysis: Use IBM SPSS Statistics 20 and GraphPad Prism

[0153] 7.0 for data processing and analysis. Whether there are differences between two groups of samples is tested by Student's t-test, and the differences between more than two groups of samples are tested by One Way ANOVA. P < 0.05 is regarded as a significant difference in the statistical results.

[0154] In this application, the inventors first proved through MTT experiments that EM-12 has a proliferation inhibitory effect on 6 ovarian cancer cells with different p53 statuses, while having less killing effect on normal ovarian epithelial cells (IOSE80). Compared with 4 p53 mutant or deleted ovarian cancer cells (TOV-112D, OVCAR-3, SK-OV-3), EM-12 has a greater killing effect on 3 p53 wild-type ovarian cancer cell lines (A2780, OVCAR-5, OVCAR-8).

[0155] Using the Drug Affinity Responsive Target Stability technology, it was found that a protein with a molecular weight of 90 kDa could be protected by EM-12 from enzymatic degradation. The molecular weight of MDM2 is 90 kDa and it is related to the p53 protein. It is speculated that the binding site of EM-12 is MDM2. Then, through molecular docking models and immunoblotting experiments, it was verified that the target of EM-12 is indeed MDM2. Next, in the blocking experiment, knocking down p53 in 3 wild-type p53 ovarian cancer cells weakened the cytotoxic effect of EM-12; in the rescue experiment, after transfecting Flag-p53-wt (wild type) into p53-deficient ovarian cancer cells, it was found that the cytotoxic effect of EM-12 was enhanced, while transfecting Flag-p53-mt (mutant type) could not enhance the cytotoxic effect of EM-12. This indicates that the inhibitory effect of EM-12 on the proliferation of ovarian cancer cells is related to the expression status of p53, and EM-12 exerts its anti-tumor effect through p53.

[0156] The results of the co-immunoprecipitation experiment showed that EM-12 promoted the dissociation of MDM2 from p53. At the same time, through immunoblotting experiments, it was found that EM-12 reduced the ubiquitination of p53, thereby prolonging the half-life of p53.

[0157] Under a laser confocal microscope, it was observed that EM-12 could promote the nuclear translocation of p53. At the same time, after separating nuclear proteins and cytoplasmic proteins, the results of immunoblotting experiments showed that EM-12 decreased the protein level of p53 in the cytoplasm and increased the protein level of p53 in the nucleus.

[0158] Through immunoblotting experiments, it was found that EM-12 inhibited the protein expression level of mTOR, and the phosphorylation levels of its downstream proteins ULK1 (Ser757) and p70s6k (Thr389) decreased, while the protein expression level of LC3-II increased, indicating that EM-12 induced autophagy activation in A2780 cells. The protein expression level of p62 increased significantly under the action of EM-12, indicating that autophagic flux was blocked. The expression of the precursor form of cathepsin L, precursor-Cathepsin L, gradually increased, while the expression of its mature form, mature-Cathepsin L, gradually decreased. Subsequently, under a laser confocal microscope, it was observed that EM-12 inhibited the formation process of autophagolysosomes, thereby blocking autophagic flux. Subsequently, we transfected SiRNA to knock down Atg5 and block autophagy, and the protein level of p53 increased significantly. These experimental results indicate that wild-type p53 can be degraded through the autophagolysosomal degradation pathway, and under the stimulation of EM-12, autophagy blockade is beneficial to increasing the protein expression level of p53.

[0159] The changes in the cell cycle were detected by flow cytometry. The results showed that EM-12 induced G1 / S phase arrest in A2780 cells. Meanwhile, the results of qRT-PCR and immunoblotting experiments showed that after treatment with EM-12, the protein levels of p53, p21, and p27 increased, the phosphorylation levels of Rb (Ser807, 795) decreased significantly, and the downstream genes of p53, p21, and Noxa also increased significantly at the mRNA level, while the mRNA level of p53 showed no obvious change. The immunoblotting results showed that EM-12 decreased the protein levels of apoptosis-related proteins Caspase-9, Caspase-3, and Caspase-8, significantly increased the expression of the pro-apoptotic protein Bax, decreased the expression of the anti-apoptotic proteins Bcl-2 and Mcl-1, and decreased the expression of PARP while increasing the expression of cleaved-PARP. The EdU experiment was further used to detect whether EM-12 had an inhibitory effect on cell proliferation. The experimental results showed that EM-12 could inhibit the proliferation of A2780 cells in a concentration-dependent manner. The flow cytometry results showed that the proportion of the FITC-cl-Caspase-3 cell population increased to 30.7% after EM-12 treated A2780 cells for 24 h. In addition, the number of Tunel-positive cells with green fluorescence staining in the cell nuclei of the Control group was less than that of the EM-12 treatment group, and it was concentration-dependent. The above experimental results indicate that EM-12 can induce G1 / S phase arrest in A2780 cells and induce apoptosis, thereby exerting an inhibitory effect on the proliferation of ovarian cancer cells.

[0160] The immunoblotting experimental results showed that EM-12 also increased the p53 protein level in other p53 wild-type ovarian cancer cells (OVCAR-5, OVCAR-8), and cleavage bands of PARP appeared, indicating that EM-12 can increase the level of p53 in tumor cells and induce apoptosis, which has universality.

[0161] In summary, the inventors found that EM-12 has an inhibitory effect on the proliferation of cancer cells with 6 different p53 statuses. Among them, the IC50 of 3 p53 wild-type ovarian cancer cell lines is the lowest, and the killing effect on normal ovarian epithelial cells IOSE80 is relatively small. EM-12 targets MDM2, promotes the dissociation of MDM2 from p53, inhibits the proteasome degradation pathway of p53. At the same time, EM-12 blocks the autophagic flux and inhibits the lysosomal degradation pathway of p53, resulting in an increase in the intracellular p53 level, which has universality. The accumulation of p53 induces G1 / S phase arrest in A2780 cells and causes apoptosis, thereby exerting an anti-tumor effect. This study clarified the anti-tumor effect and its molecular mechanism of the novel sesquiterpene lactone compound EM-12 extracted from Elephantopus scaber L. in p53 wild-type ovarian cancer cells, providing an experimental basis for the future clinical application of EM-12 in ovarian cancer treatment.

[0162] The above-described embodiments merely represent several embodiments of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patented application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. Use of a sesquiterpene lactone compound in the preparation of a drug for treating or adjuvant treating p53 wild-type ovarian 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. The use according to claim 1, characterized in that: The drug includes the following properties: targeting MDM2 and inhibiting the proteasomal degradation of p53.

3. The use according to claim 2, characterized in that: The drug also includes the following characteristics: arresting the p53 wild-type ovarian cancer cells in the G1 / S phase of the cell cycle.

4. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the drug is selected from: injection, tablet, capsule, kit or patch.

5. Use of a sesquiterpene lactone compound in the preparation of a drug for inhibiting the proliferation of p53 wild-type ovarian 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:

6. The use according to claim 5, characterized in that: The p53 wild-type ovarian cancer cells are: A2780, OVCAR-5 and OVCAR-8.

7. Use of a sesquiterpene lactone compound in the preparation of an inhibitor for inducing apoptosis in p53 wild-type ovarian 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:

8. Use of a sesquiterpene lactone compound in the preparation of an anti-p53 wild-type ovarian 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:

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