Application of acetyl alkannin or pharmaceutically acceptable salt thereof in preparation of antitumor drugs

Abstract

The invention discloses an application of acetyl alkannin or pharmaceutically acceptable salts thereof in preparation of antitumor drugs, and belongs to the technical field of medicines, and tumors comprise liver cancer or lung cancer. Acetyl alkannin can inhibit ATM / CHK-2 pathway, induce DNA damage of Huh-7 cells and A549 cells, and promote cell apoptosis.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technologies, and particularly to the use of acetylakannin or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs. Background Art

[0002] Cancer has become one of the major global public health problems, seriously threatening human health. It can occur in various parts of the human body, destroying the structure and function of normal tissues and organs, causing complications such as pain and bleeding, and affecting the quality of life and survival period of patients.

[0003] Cancer treatment often requires comprehensive means such as surgery, radiotherapy and chemotherapy. The process is long and accompanied by many side effects, such as nausea, vomiting, hair loss, and decreased immunity, bringing great physical and mental pain to patients. From a social perspective, the high incidence and mortality of cancer increase the burden on medical resources, consuming a large amount of manpower, material resources and financial resources for treatment and care. Moreover, the loss of the labor force affects social and economic development and reduces the level of social productivity. The research and development of anti-tumor drugs is an important way to treat cancer. Summary of the Invention

[0004] The main object of this application is to provide the use of acetylakannin or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs.

[0005] This application provides the use of acetylakannin or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs, wherein the tumors include liver cancer or lung cancer.

[0006] In some embodiments of this application, the anti-tumor drug further includes cisplatin.

[0007] In some embodiments of this application, the acetylakannin or a pharmaceutically acceptable salt thereof is used as a resistance reversal agent in anti-tumor drugs.

[0008] In some embodiments of this application, the acetylakannin or a pharmaceutically acceptable salt thereof is used as a drug sensitizer in anti-tumor drugs.

[0009] In some embodiments of this application, the pharmaceutically acceptable salts include salts formed with inorganic acids, organic acids, amino acids, alkali metals or alkaline earth metals.

[0010] In some embodiments of this application, the raw materials for preparing the anti-tumor drug further include pharmaceutically acceptable excipients;

[0011] and / or, the pharmaceutically acceptable excipients include at least one of diluents, excipients, binders, wetting agents, lubricants, disintegrants, absorption promoters, surfactants, adsorption carriers, sweeteners and flavoring agents.

[0012] In some embodiments of the present application, the dosage forms of the anti-tumor drug include at least one of injection, tablet, patch, suspension, granule, capsule, powder, emulsion, solution, pill, dripping pill, oral preparation, suppository, enema, aerosol or drop.

[0013] In some embodiments of the present application, the use of acetylakannin or a pharmaceutically acceptable salt thereof as a cell viability inhibitor in an anti-tumor drug is provided.

[0014] In some embodiments of the present application, the use of acetylakannin or a pharmaceutically acceptable salt thereof as a cell apoptosis promoter in an anti-tumor drug is provided.

[0015] In some embodiments of the present application, acetylakannin or a pharmaceutically acceptable salt thereof inhibits the expression of ATM in tumor cells.

[0016] The beneficial effects of the embodiments of the present application are as follows: Acetylakannin can inhibit the ATM / CHK-2 pathway, induce DNA damage in Huh-7 cells and A549 cells, and promote cell apoptosis. Therefore, it can be used for the preparation of anti-tumor drugs, especially for the treatment of liver cancer or lung cancer, providing a reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present drawings or related technologies, the drawings required for use in the description of the embodiments or related technologies will be briefly introduced below.

[0018] Figure 1 It is a screening result diagram of the mRNA expression data set of ATM and RAD51 in liver cancer and lung cancer solid tumors;

[0019] Figure 2 It is a result diagram of the expression levels of ATM and downstream pathway proteins detected by Western blot experiment under the action of DDP with different concentrations and different action durations, and the result diagram of the influence of DDP with different concentrations on cell proliferation detected by colony formation experiment and EdU experiment;

[0020] Figure 3 It is a result diagram of the influence of KU-55933 on the cell viability of A549 and Huh-7 cells detected by MTT experiment, the influence of the combination of DDP and KU-55933 on the expression levels of ATM and downstream pathway proteins detected by Western blot experiment, and the result diagram of the influence of the combination of DDP and KU-55933 on cell proliferation detected by colony formation experiment and EdU experiment;

[0021] Figure 4The ATM was knocked down by transfecting A549 cells with siRNA sequences, and the test results were verified by Western blot experiments. The results of the Annexin V-FITC / PI double staining experiment for detecting the number of apoptotic cells after knocking down ATM and the results of the colony formation experiment for detecting the effect of knocking down ATM on cell viability;

[0022] Figure 5 The figure shows the results of the MTT experiment for detecting the effects of shikonin and its six analogues on the cell viability of A549 and Huh-7 cells, and the results of the Western blot experiment for detecting the effects of shikonin and six analogues on the expression levels of ATM and p-ATM in A549 cells and Huh-7 cells;

[0023] Figure 6 The figure shows the results of the MTT experiment for detecting the effects of the combined action of acetylakannin and DDP on cell viability, the results of the Western blot experiment for detecting the effects of the combination of DDP and acetylakannin on the expression levels of ATM and downstream pathway proteins, and the results of the colony formation experiment and EdU experiment for detecting the effects of the combination of DDP and acetylakannin on cell proliferation;

[0024] Figure 7 The figure shows the results of Western blots experiments for detecting the effect of acetylakannin on the expression level of ATM in A549 cells, the effect of acetylakannin on the half-life of ATM in A549 cells, and the specific molecular mechanism of acetylakannin inhibiting ATM, and the results of the CETSA experiment for detecting the effect of acetylakannin on the thermal stability in A549 cells.

[0025] The realization, functional characteristics and advantages of the purpose of this application will be further described with reference to the embodiments and the accompanying drawings. Detailed Description of the Embodiments

[0026] Hereinafter, the embodiments of the application of acetylakannin or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs, which specifically disclose this application, will be described in detail with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where the detailed descriptions of well-known matters and the repeated descriptions of actually identical structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided for those skilled in the art to fully understand this application, and are not intended to limit the subject matter recited in the claims.

[0027] Acetylakannin is a naphthoquinone compound and also an analogue of shikonin, with the chemical molecular formula C 18 H 18 O6, and the chemical structural formula is as follows:

[0028]

[0029] Previous related studies have found that shikonin is a broad-spectrum DNA damage repair (DDR) inhibitor, which enhances the anti-tumor effect of chemotherapeutic drugs by inhibiting ATM. In view of its problems such as poor solubility, poor chemical stability and non-selective toxicity, a variety of analogs have been developed. As a shikonin analog, acetylakannin has a powerful anti-tumor effect, but its molecular mechanism of anti-hepatocellular carcinoma and lung cancer is still unclear.

[0030] On the other hand, DDP is a cornerstone drug for the chemotherapy of hepatocellular carcinoma and lung cancer, but more and more chemoresistance occurs, leading to the recurrence of hepatocellular carcinoma and lung cancer. In this regard, finding a new drug with low toxicity and side effects or a new drug that can enhance the sensitivity of tumor cells to chemotherapeutic drugs is of great significance for the clinical treatment of hepatocellular carcinoma and lung cancer.

[0031] Based on this, the embodiments of the present application provide the use of acetylakannin or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug, and the tumor includes hepatocellular carcinoma or lung cancer.

[0032] Acetylakannin can inhibit the ATM / CHK-2 pathway, induce DNA damage in Huh-7 cells and A549 cells, and promote apoptosis.

[0033] In some embodiments of the present application, the anti-tumor drug further includes cisplatin (DDP).

[0034] In some embodiments of the present application, the use of acetylakannin or a pharmaceutically acceptable salt thereof as a drug resistance reverser in an anti-tumor drug.

[0035] In some embodiments of the present application, the use of acetylakannin or a pharmaceutically acceptable salt thereof as a drug sensitizer in an anti-tumor drug.

[0036] DDP can activate the downstream ATM pathway in hepatocellular carcinoma and lung cancer cells, promote the repair of tumor cell DNA damage, and lead to the generation of chemoresistance. Acetylakannin can inhibit the ATM pathway and promote apoptosis after combination with cisplatin, playing a role in sensitizing cisplatin.

[0037] In some embodiments of the present application, the use of acetylakannin or a pharmaceutically acceptable salt thereof as a cell viability inhibitor in an anti-tumor drug.

[0038] In some embodiments of the present application, the use of acetylakannin or a pharmaceutically acceptable salt thereof as a cell apoptosis promoter in an anti-tumor drug.

[0039] In some embodiments of the present application, acetylakannin or a pharmaceutically acceptable salt thereof inhibits the expression of ATM in tumor cells.

[0040] Acetylakannin has a high inhibitory effect on the viability of Huh-7 cells, and the IC 50 at 48 h is only 5.86 μM. At the same concentration, the inhibitory effect of acetylakannin on the viability of Huh-7 and A549 cells is much more significant than that of the conventional chemotherapeutic drug cisplatin. Acetylakannin has a strong binding affinity with ATM in A549 cells and promotes the degradation of ATM in A549 cells through a caspase-dependent pathway, thereby shortening the half-life of intracellular ATM.

[0041] In some embodiments of the present application, pharmaceutically acceptable salts include salts formed with inorganic acids, organic acids, amino acids, alkali metals or alkaline earth metals.

[0042] In some embodiments of the present application, the inorganic acids include at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, and hydrobromic acid.

[0043] In some embodiments of the present application, the organic acids include at least one of 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, sulfanilic acid, 2-acetoxybenzoic acid, and hydroxyethanesulfonic acid.

[0044] In some embodiments of the present application, the alkali metals include at least one of lithium, sodium, and potassium.

[0045] In some embodiments of the present application, the alkaline earth metals include at least one of calcium and magnesium.

[0046] In some embodiments of the present application, the amino acids include lysine.

[0047] In some embodiments of the present application, the raw materials for preparing the anti-tumor drug further include pharmaceutically acceptable excipients.

[0048] In some embodiments of the present application, pharmaceutically acceptable excipients include at least one of diluents, excipients, binders, wetting agents, lubricants, disintegrants, absorption promoters, surfactants, adsorption carriers, sweeteners, and flavoring agents.

[0049] In some embodiments of the present application, the excipients include at least one of lactose, polyethylene glycol, sodium citrate, dicalcium phosphate, and water.

[0050] In some embodiments of the present application, the binders include at least one of cellulose derivatives, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic.

[0051] In some embodiments of the present application, the cellulose derivative includes carboxymethyl cellulose.

[0052] In some embodiments of the present application, the alginate includes alginate.

[0053] In some embodiments of the present application, the wetting agent includes glycerol.

[0054] In some embodiments of the present application, the disintegrant includes at least one of agar, calcium carbonate, sodium bicarbonate, potato starch, tapioca starch, alginic acid, silicate, and sodium carbonate.

[0055] In some embodiments of the present application, the absorption promoter includes quaternary ammonium compounds.

[0056] In some embodiments of the present application, the surfactant includes cetyl alcohol.

[0057] In some embodiments of the present application, the adsorption carrier includes at least one of kaolin, bentonite, and saponite.

[0058] In some embodiments of the present application, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, sodium dodecyl sulfate, and polyethylene glycol.

[0059] In some embodiments of the present application, the dosage forms of the anti-tumor drug include at least one of injection, tablet, patch, suspension, granule, capsule, powder, emulsion, solution, pill, dripping pill, oral preparation, suppository, enema, aerosol, or drops.

[0060] The experimental materials and methods adopted in the embodiments of the present application are described below.

[0061] Cell lines: The human hepatocellular carcinoma cell line Huh-7 and the human lung cancer cell line A549 were both purchased from the American Type Culture Collection (ATCC).

[0062] Methods: MTT assay was used to detect the effects of DDP, acetylakannin, shikonin and its analogues on the viability of hepatocarcinoma and lung cancer cells. EdU and cell colony formation assays were used to detect the effect of acetylakannin combined with DDP on the proliferation ability of human hepatocellular carcinoma cell Huh-7. Flow cytometry and immunoblotting were used to detect the effects of acetylakannin on cell apoptosis and the ATM pathway. CETSA assay was used to detect the effect of acetylakannin on the thermal stability of ATM.

[0063] Example 1: MTT assay.

[0064] (1) Experimental design: a zero - adjustment group, a control group, and a drug - adding group. Only the culture medium is added to the zero - adjustment group. The drug - adding group is added with the culture medium, cells, and the experimental drug. The control group is added with the volume of DMSO in the maximum drug - concentration group. Five replicate wells are set in each group. The specific drug concentrations set in each group can be referred to as shown in the respective figures.

[0065] (2) When the cells are cultured to the logarithmic growth phase, the cells are digested with trypsin digestion solution containing 0.25% EDTA, and the digestion is terminated with the culture medium containing 10% FBS and blown into a single - cell suspension. Then the cells are collected by low - speed centrifugation at 800 rpm for 3 min. After resuspending the cells with the culture medium, the cell density is calculated, and 100 μL of the cell suspension is seeded into a 96 - well plate at a density of 4000 - 6000 cells per well, and then cultured adherently overnight. The outer perimeter of the well plate is filled with 200 μL of 1×PBS to reduce the evaporation of the culture medium.

[0066] (3) Using the serial dilution method, the drug is diluted to twice the experimental concentration. 100 μL of the drug is added to the cells, so that the drug concentration is diluted by half to the experimental working concentration. After culturing for 48 h, 20 μL of MTT solution (5 mg / mL) is added to each well of the zero - adjustment group, the control group, and the experimental group, and then cultured for another 4 - 6 h. The mixed solution of the culture medium and MTT is aspirated completely with a vacuum suction pump, and then 150 μL of DMSO solution is added to each well, and shaken at low speed for 10 min. After the formazan purple crystals are fully dissolved, the absorbance at 570 nm is detected using a multi - functional microplate reader, and the cell survival rate of each group is calculated by calculating the average absorbance value of each group.

[0067] (4) Cell survival rate (%)=(average absorbance value of the experimental group - average absorbance value of the zero - adjustment group) / (average absorbance value of the control group - average absorbance value of the zero - adjustment group)×100%. IBM SPSS Statistics 20 is used to calculate the IC50 (Mean±SD) and GraphPad Prism 7.0 is used for plotting.

[0068] Example 2: Detection of cell proliferation by the EdU method.

[0069] (1) When the cells are cultured to the logarithmic growth phase, after digestion, the cells are centrifuged and collected, and after counting, 100 μL of the cell suspension is seeded into a 96 - well plate at a density of 5000 cells per well. Three replicate wells are set in each group and cultured overnight.

[0070] (2) After the cells adhere to the wall, the culture medium is aspirated, and different concentrations of drugs DDP (A549: 0, 5, 10, 20 μM; Huh - 7: 0, 2, 4, 8 μM), KU - 55933 (10 μM), and acetylakannin (1 μM) are added to each well of different cells, and the drug treatment is continued for 24 h.

[0071] (3) After aspirating the culture medium, add 100 μL of fresh culture medium containing 10 μM EdU working solution and continue to incubate for 2.5 h.

[0072] (4) After aspirating the EdU labeling solution, add 100 μL of 4% paraformaldehyde to fix for 20 min. After removing the fixing solution, add 100 μL of 2 μg / μL glycine solution and continue to incubate at room temperature for 5 min to remove the residual 4% paraformaldehyde.

[0073] (5) Aspirate the glycine solution, add 100 μL of 3% BSA solution to wash 1 - 2 times, then add 100 μL of 0.5% Triton X - 100 and incubate at room temperature for 15 min.

[0074] (6) Aspirate 0.5% Triton X - 100 and wash the cells with 3% BSA 1 - 2 times, then add 100 μL of Click - iT reaction mixture prepared according to the reagent instruction manual and incubate at room temperature in the dark for 30 min.

[0075] (7) Remove the reaction mixture, add 100 μL of 1×PBS to each well to wash 1 - 2 times, and add 100 μL of 5 μg / mL Hochest33342 solution and incubate at room temperature in the dark for 15 min. Then wash with 100 μL of 1×PBS 1 - 2 times and add 50 μL of anti - fluorescence quenching agent.

[0076] (8) Perform photographing and analysis under an inverted fluorescence microscope. kFluor488: Ex / Em = 495 nm / 519 nm, green fluorescence. Hoechst33342: Ex / Em = 350 nm / 461 nm, blue fluorescence.

[0077] Example 3: Annexin V - FITC / PI double staining for detecting cell apoptosis.

[0078] (1) When the cells are cultured to the logarithmic growth phase, digest and centrifuge to collect the cells. After counting, seed 3.0×10⁵ cells in each well of a 6 - well plate and continue to culture overnight.

[0079] (2) After the cells adhere to the wall, aspirate the culture medium, add different concentrations of drugs: siATM1 (100 pmol), siNC (100 pmol), DDP (4 μM) or combined drugs, and continue to culture for 48 h.

[0080] (3) Digest and collect cells with trypsin without EDTA to avoid false positives caused by the binding of EDTA to Annexin V. After the cells are washed twice with pre-cooled 1×PBS, first add 300 μL of Bind Buffer to the sample to resuspend the cells, then add 3 μL of Annexin V-FITC, mix well and incubate in the dark at room temperature for 15 min. PI staining: Add 3 μL of PI staining solution, gently pipette to mix the cells, and incubate in the dark at room temperature for 15 min. Then detect the apoptosis of the sample by flow analyzer.

[0081] Example 4: Immunoblotting.

[0082] (1) Protein quantification

[0083] Take 3.0×10 5 cells in good growth state and seed them in a 6-well plate. After overnight adherent culture, add different concentrations of drugs: DDP (4 μM or 6 μM), KU-55933 (10 μM), siATM1 (100 pmol), acetylakannin treatment (1 μM), CHX (10 μM), MG132 (1 μM), BafA1 (400 nM), Z-VAD-FMK (20 μM) and treat for 24 h.

[0084] After the drug treatment is completed, aspirate the culture medium, add pre-cooled 1×PBS for rinsing, and then add 100 - 200 μL of RIPA strong lysis buffer on ice. Protease inhibitor PMSF and protein phosphatase inhibitor mixture are pre-added to the RIPA strong lysis buffer. After lysing on ice for half an hour, collect the cell lysate into a nuclease-free microcentrifuge tube. Pre-open the high-speed refrigerated centrifuge and separate the protein supernatant by centrifugation at 4°C, 12,000 rpm for 15 min. Transfer the protein supernatant to a new nuclease-free microcentrifuge tube and determine the protein concentration by BCA kit.

[0085] After protein quantification, add SDS-PAGE protein loading buffer (6×) to dilute it to 1×, and heat it in a metal bath at 95°C for 5 min. Aliquot the protein supernatant and store it at -80°C.

[0086] (2) Electrophoresis; Prepare a polyacrylamide gel with an appropriate concentration according to the molecular weight of the target protein to be detected. 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 Rainbow protein Marker with the molecular weight of the target protein runs fully apart.

[0087] (3) Transfer the membrane; after electrophoresis, cut off the excess gel part and place 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 cassette. After filling it with the electrotransfer solution, transfer the membrane for 90 min with a constant current of 200 mA. Fill the outer periphery of the transfer tank with ice cubes.

[0088] (4) Blocking; after membrane transfer, soak the membrane in 5% blocking milk, shake it slowly, and incubate it at room temperature for 1 h.

[0089] (5) Antibody incubation and chemiluminescence imaging; after blocking, wash off the excess milk particles with 1×TBS buffer. The primary antibody is diluted 1:1000 with the primary antibody diluent and then the membrane is soaked in the primary antibody and incubated overnight at 4°C. After the primary antibody incubation, recover the primary antibody and add 1×TBST washing solution. Set the shaker at 130 rpm and wash the membrane for 10 min each time, for a total of 3 times. After washing the membrane, add 1×TBST washing solution, set the shaker at 130 rpm, wash the membrane for 10 min each time, for a total of 3 times. After washing the membrane, apply the ultrasensitive ECL chemiluminescence solution on the membrane, expose it through a chemiluminescence imager, and collect and analyze the experimental results.

[0090] (6) Statistics and analysis:

[0091] Data processing and analysis are performed using IBM SPSS Statistics 20 and GraphPad Prism 7.0. Whether there are differences between the two groups of samples is tested by Student's t-test, and the comparison of more than two groups of samples is tested for inter-group differences by One Way ANOVA. P < 0.05 is regarded as a significant difference in the statistical results.

[0092] Example 5: Cell colony formation assay.

[0093] (1) Cell culture: Take cells in the logarithmic growth phase and evenly inoculate about 500 - 1000 cells per well into a 6-well plate, and observe under the microscope to ensure that the cells are evenly seeded in the 6-well plate.

[0094] (2) Drug treatment: After the cells adhere to the wall, remove the culture medium, and add 2 mL of drug-containing culture medium DDP (2 μM), KU-55933 (5 μM), acetylakannin (1 μM), siATM1 (100 pmol), siNC (100 pmol) to each well to treat the cells, and continue to culture for 7 - 14 d.

[0095] (3) Imaging and analysis: After the diameter of the cell clones reached 0.3 mm to 1 mm, the culture medium was removed, and the cells were washed 2 - 3 times with PBS. An appropriate amount of paraformaldehyde solution was added to each well to completely cover the cells. After fixation at room temperature for 30 min, the fixing solution was removed and the cells were washed with PBS. The 1% crystal violet staining solution was diluted to 0.1% with PBS, and 1 mL was added to each well for 30 min of staining. The staining solution was removed, and then the well plate was gently rinsed with running water. After air-drying, pictures were taken or scanned and saved. 30% glacial acetic acid was added to completely dissolve the crystal violet. Then, the solution in each well was transferred to three replicate wells of a 96-well plate, and the absorbance OD value at a wavelength of 450 nm was read on an enzyme-linked immunosorbent assay (ELISA) reader, and graphs were plotted using GraphPad Prism 9.1.1.

[0096] Example 6: siRNA transfection experiment.

[0097] Take A549 cells in good growth state (2×10 5 cells per well) and seed them in a 6-well plate, and culture them in an environment without 1% penicillin-streptomycin.

[0098] According to the transfection reagent instructions, the siRNA solution and the liposome solution LipofectamineTM 2000 were diluted separately with low-serum medium Opti-MEM. Subsequently, the diluted siRNA solution was added dropwise to LipofectamineTM 2000, gently mixed, and left standing at room temperature for 20 min to form a transfection complex.

[0099] Remove the cell culture supernatant, and wash the wells of the plate 2 times with Opti-MEM. After mixing the transfection complex with Opti-MEM, continue to co-culture with the cells for 4 to 6 h. Finally, replace the culture supernatant with normal medium for subsequent processing.

[0100] The sequences of the siATM used are as follows:

[0101] Sequence 1 is 5’-GAAAGAGAATGGATTAGAA-3’;

[0102] Sequence 2 is 5’-GCAACATACTACTCAAAGA-3’;

[0103] Sequence 3 is 5’-GAATGAAGATTCCAACATA-3’.

[0104] Example 7: Cellular thermal shift assay (CETSA).

[0105] A549 cells in logarithmic growth phase were seeded in 6-well plates. After treatment with acetylakannin (10 μM) or solvent (DMSO) for 24 h, cell lysis was performed with RIPA lysis buffer containing PMSF and phosphatase inhibitors for 30 min. Subsequently, the cell lysate was centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was heated at different temperatures (37, 42, 45, 47, 48, 48.5, 49, 50, 55, and 65 °C) for 3 min. After cooling with liquid nitrogen for 3 min, it was thawed at room temperature and freeze-thawed three times. Then, it was centrifuged again at 12,000 rpm for 15 min at 4 °C, and the obtained heated lysate was used for Western blot analysis.

[0106] The experimental results obtained from the above Examples 1 to 7 are analyzed and described below.

[0107] To study the mRNA expression levels of ATM and RAD51 in lung cancer and liver cancer, mRNA expression datasets of lung cancer and liver cancer were obtained from The Cancer Genome Atlas (TCGA) database, and were preprocessed and screened to obtain the results as Figure 1 shown. Figure 1 The left subgraphs in A-D in the figure are the relative mRNA expression levels of ATM or RAD51 between hepatocellular carcinoma (LIHC, 50 adjacent tissues and 369 tumor tissues) and lung adenocarcinoma (LUAD, 59 adjacent tissues and 483 tumor tissues) and their normal tissues in the TCGA database; Figure 1 The right subgraphs in A-D in the figure are paired box plots of ATM or RAD51 expression between paired normal tissues and LIHC (n = 50) or LUAD (n = 59) tumor tissues in the TCGA dataset. As Figure 1 shown in the results of A-D in the figure, compared with normal tissues, the expressions of ATM and RAD51 were significantly upregulated in liver cancer and lung cancer tissues (50 and 59 adjacent tissues, 369 and 483 tumor tissues respectively). In addition, in the paired comparisons of hepatocellular carcinoma (LIHC) with adjacent tissues (n = 50) or lung adenocarcinoma (LUAD) with adjacent tissues (n = 59), ATM and RAD51 also showed significantly higher expressions (right group, Figure 1 A-D).

[0108] These results indicate that the expressions of ATM and RAD51 are upregulated in liver cancer and lung cancer, suggesting that they may be potential biomarkers for predicting tumor malignant progression.

[0109] Cisplatin exerts its cytotoxic effect by inducing DNA damage in tumor cells, and ATM is an upstream protein in the DNA damage response pathway. Therefore, in order to determine the effect of DDP on ATM and its downstream DDR pathway in non-small cell lung cancer A549 cell line and hepatocellular carcinoma Huh-7 cells, Western blot experiments were used to detect the expression levels of ATM and downstream pathway proteins under the action of DDP at different concentrations and different action times. The results are as follows: Figure 2 The results are shown. Figure 2 A and B show that DDP enhanced the expression levels of p-ATM and its downstream proteins p-CHK2, CtIP, and RAD51 in a dose- and time-dependent manner.

[0110] It can also be found that after DDP treatment, Figure 2 As shown in A and B, the expression level of cl-PARP increased. The effect of DDP on the survival and proliferation of A549 and Huh-7 cells was evaluated by clone formation assay, and the results were consistent with those of Western blot. Figure 2 In C, Figure 2 D is the statistical result of C. DDP reduced the colony formation ability of A549 and Huh-7 cells in a dose-dependent manner. To further prove this, the EdU experiment was used to detect whether DDP had a significant cell proliferation inhibitory effect. Figure 2 As shown in E, after 48 h of DDP treatment, the number of tumor cell proliferation was significantly reduced. The above experimental results show that DDP can activate ATM and promote the increase of downstream DDR pathway protein levels, while inducing tumor cell apoptosis.

[0111] To further clarify whether DDP promotes tumor cell homeostasis by activating ATM and downstream DDR pathways, thereby leading to chemotherapy resistance, the MTT assay was used to detect the effect of ATM inhibitor KU-55933 on the viability of A549 and Huh-7 cells. Figure 3 The results are shown. Figure 3 As shown in A and B, compared with DDP monotherapy, the combination therapy effectively reduced cell viability, IC 50 The levels of KU-55933 and DDP decreased from 49.54 μM to 26.76 μM and 23.55 μM to 9.25 μM, respectively. Subsequently, Western blot experiments were performed to detect the effects of KU-55933 and DDP on ATM phosphorylation and its downstream factors. Figure 3As shown in C and D, KU-55933 inhibited ATM phosphorylation induced by DDP in A549 and Huh-7 cells, and also had a certain inhibitory effect on CHK2 phosphorylation. At the same time, the combination treatment promoted the cleavage of PARP, indicating that inhibiting DDP-induced ATM activation could lead to apoptosis of A549 and Huh-7 cells.

[0112] To further confirm the above conclusion, the EdU experiment was used to detect the effect of the combination treatment on the proliferation of A549 and Huh-7 cells. As Figure 3 shown in E, compared with DDP alone, the combination of KU-55933 and DDP significantly reduced the proliferation of A549 and Huh-7 cells. Consistent with the EdU experiment, in Figure 3 F, it was visible that the combination treatment group significantly reduced colony formation. The above results indicate that inhibiting ATM activation can reverse the downstream activation of DDR induced by DDP, thereby inhibiting tumor cell proliferation and promoting apoptosis.

[0113] To further confirm the role of ATM in DDP-induced apoptosis, referring to the results in Figure 4 , three siRNA sequences of ATM (siATM1, siATM2, siATM3) were transfected into A549 cells to knockdown ATM, and then the three sequences were verified by Western blot experiment. As Figure 4 shown in A, the siATM1 sequence had the best knockdown effect. Therefore, siATM1 was selected for subsequent experiments. As Figure 4 shown in B, when ATM in A549 cells was knocked down by siRNA, the protein levels of p-ATM and its downstream effectors such as p-CHK2, CtIP, and RAD51 were inhibited. After treatment with DDP, the expression levels of ATM and downstream proteins remained low. It is worth noting that after A549 cells with knocked-down ATM were treated with DDP, the expression level of cl-PARP increased, that is, knocking down ATM induced apoptosis.

[0114] To verify the above Western blot results, A549 cells were double-stained with Anniexin V-FITC / PI and the results were analyzed by flow cytometry, and the results shown in Figure 5 were obtained. Figure 5 The results in C showed that compared with the DDP single-treatment group, the combination treatment after knocking down ATM could significantly promote the apoptosis of A549 cells. In the Figure 4 colony formation experiment in D, similar results were also seen. The combination of siATM and DPP could significantly inhibit the proliferation ability of A549 cells. Combining the above experimental results, the conclusion was that knocking down ATM with siRNA would increase the sensitivity of A549 cells to DDP.

[0115] At present, a large number of relevant research reports on the anti-cancer activity of shikonin have been published. It has been reported that shikonin analogs have advantages such as better solubility and chemical stability, but these analogs have not been systematically studied. Therefore, the MTT method was used to evaluate the effects of shikonin and its six analogs on the cell viability of A549 and Huh-7 cells, referring to Figure 5 the results shown, Figure 5 In the table shown, lithospermic acid, shikonin cyanide, shikonofuran A, acetylakannin, β-acetoxyisovaleryl akannin, β,β-dimethylacryloyl shikonin, and shikonin are listed from top to bottom in order. As Figure 5 The results of A and B in

[0116] showed that in A549 and Huh-7 cells, acetylakannin showed stronger anti-tumor effects compared to the other five compounds. By measuring the IC50 values in A549 and Huh-7 cells, it was found that the half-maximal inhibitory concentrations of A549 and Huh-7 cells were relatively low, 4.93 μM and 5.81 μM, respectively. Figure 5 The results shown in C of

[0117] showed that under the induction of DDP, acetylakannin significantly inhibited the phosphorylation of ATM in A549 and Huh-7 cells. At the same time, after exposure to acetylakannin, a decrease in the expression level of ATM was also observed in A549 cells, but this change was not observed in Huh-7 cells. In summary, the above experimental results indicate that acetylakannin has the functions of inhibiting the cell viability of A549 and Huh-7 cells and inhibiting the phosphorylation of ATM. Figure 6 the results shown. As Figure 6 shown in A of 20 , the combined use of acetylakannin (IC 50 ) and DDP significantly reduced the cell viability of A549 and Huh-7 cell lines, reducing the IC50 of A549 from 49.54 μM to 2.20 μM and the IC Figure 6In B, compared with the use of DDP alone, A549 and Huh-7 cells showed higher sensitivity to the co-treatment of acetylakannin and DDP, that is, the number of clones was significantly reduced. The results of the EdU staining experiment were consistent with the colony formation experiment. As Figure 6 shown in C, the proportion of the number of newborn cells in the DDP combined with acetylakannin group was significantly reduced compared with the DDP single drug group. The results of both the colony formation experiment and the EdU experiment proved that acetylakannin combined with DDP could significantly inhibit the proliferation of A549 and Huh-7 cells.

[0118] To further investigate the effect of acetylakannin on the levels of ATM and downstream proteins in A549 and Huh-7 cells, Western blot was used to detect the effect of acetylakannin and DDP on the phosphorylation levels of ATM and its downstream factors. As Figure 6 shown in D, acetylakannin inhibited the phosphorylation of ATM and CHK2 in A549 and Huh-7 cells induced by DDP. Downstream factors of ATM such as CtIP and RAD51 were also inhibited by the combination treatment. At the same time, the expression level of cleaved-PARP was also significantly increased, indicating that acetylakannin combined with DDP could promote the apoptosis of A549 and Huh-7 cells. The above experimental results showed that acetylakannin could inhibit ATM activation and overcome the chemoresistance of A549 and Huh-7 cells to DDP.

[0119] In previous experiments, it has been confirmed that acetylakannin can inhibit the phosphorylation of ATM. To further prove whether the inhibition of ATM activation by acetylakannin is dose-dependent, Western blots were used to detect the changes in the expression level of ATM in A549 cells, and the Figure 7 results were obtained. As Figure 7 shown in A, as the drug concentration of acetylakannin increased, the ATM protein level showed an obvious downward trend. Therefore, it was speculated that acetylshikonin might target ATM. To prove the above conjecture, a CETSA experiment was carried out. The CETSA technique is based on the principle that ligand binding enhances the thermal stability of the target protein and is widely used to verify the binding activity of drugs to their target proteins in cells. As Figure 7 shown in the results of B, compared with the DMSO group, acetylakannin increased the thermal stability of the ATM protein, which also indicated that there was an interaction between acetylakannin and ATM. In addition, as Figure 7 shown in C, molecular docking simulation showed that the affinity score of acetylakannin and the ATM protein was -7.993 kcal / mol and formed a conventional hydrogen bond: the distances from Cys2770 were and and a distance of The conventional hydrogen bond is connected to Glu2768, further proving the strong binding interaction between acetylakannin and ATM protein.

[0120] Next, the hypothesis was proposed again: acetylakannin reduces its protein level by promoting ATM degradation, and the following verification was carried out for this purpose. First, to determine whether acetylakannin affects the half-life of ATM, CHX was added to A549 cells to inhibit ATM protein synthesis. After 24 h of CHX treatment, the ATM protein level began to decrease significantly. Under the same conditions, after adding acetylakannin, the half-life of ATM was relatively shortened, as Figure 7 shown in D. Second, to further clarify the specific mechanism of acetylakannin promoting ATM degradation, inhibitors of three major degradation pathways were used in combination: the proteasome pathway inhibitor MG132, the autophagy inhibitor BafA1, and the caspase pathway inhibitor Z-VAD-FMK. As Figure 7 shown by the Western blot results in E, both BafA1 and Z-VAD-FMK showed certain inhibitory effects on ATM degradation, and the inhibitory effect of Z-VAD-FMK was the most significant. However, MG132 had little effect on acetylakannin-induced ATM degradation. These data indicate that in the presence of acetylakannin, the increase in ATM degradation is mainly achieved through the caspase pathway.

[0121] The above experimental results show that acetylakannin has a strong binding affinity with ATM in A549 cells and promotes the degradation of ATM in A549 cells through a caspase-dependent pathway, thereby shortening the half-life of intracellular ATM.

[0122] In this application, first, it was explored whether acetylakannin can effectively enhance the sensitivity of A549 and Huh-7 cells to DDP, and the mechanism of the synergistic effect between acetylakannin and DDP was revealed. The research data show that the activation of ATM and its downstream proteins and the initiation of the DNA damage response (DDR) play a key role in inducing tumor cell resistance to DDP. The research results show that acetylakannin can enhance the sensitivity of A549 and Huh-7 cells to DDP by inhibiting ATM. Next, the molecular mechanism of acetylakannin inhibiting ATM was further verified.

[0123] Platinum drugs, including cisplatin, carboplatin, oxaliplatin, etc., are one of the most widely used drugs in the chemotherapy of malignant tumors. They have cytotoxic effects on various solid tumors such as cervical cancer, colon cancer, lung cancer, breast cancer, liver cancer, ovarian cancer, nasopharyngeal cancer, etc. However, while promoting apoptosis, these drugs also activate the downstream DDR pathway, helping cells restore homeostasis and evade apoptosis, thus generating drug resistance. Taking DDP as an example, DDR is one of the mechanisms of its chemotherapy resistance, and the activated DDR pathway includes homologous recombination repair (HR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER), and mismatch repair (MMR). HR repair mainly promotes DDR by directly activating the ATM-related pathway and its downstream proteins such as CHK2, CtIP, and RAD51.

[0124] In various solid tumor cells, a series of downstream survival signaling pathways mediated by abnormal activation of ATM play a crucial role in chemotherapy resistance. Through bioinformatics analysis of the TCGA database in this study, it was found that the ATM and RAD51 genes were upregulated in liver cancer and lung cancer. Further survival analysis showed that the high expression of ATM and RAD51 led to a shortened overall survival of liver cancer patients. These results suggest that ATM and RAD51 may be related to the malignant progression of solid tumors ( Figure 1 ). In in vitro experiments, A549 lung cancer cells and Huh-7 hepatocellular carcinoma cells were stimulated with DDP, and it was observed that apoptosis of tumor cells could be induced whether stimulated under a concentration gradient or a time gradient ( Figure 2 Figures A and B). Colony formation assays and EdU assays further confirmed that DDP could inhibit the proliferation of tumor cells ( Figure 2 Figures C - E). At the same time, DDP activated compensatory signaling pathways, including the activation of ATM and its downstream CHK2 protein. In addition, ATM kinase is a key mediator in the DNA damage response. Therefore, the activation of ATM led to the upregulation of proteins related to its downstream HR signaling pathway, including CtIP and RAD51 ( Figure 2 Figures A and B). These results indicate that as a DNA-damaging agent, the cytotoxic effect of DDP on DNA may be partially offset by the cell survival mechanism, which involves the activation of the ATM-mediated DNA damage repair pathway. This mechanism plays an important role in the chemotherapy resistance of tumor cells. To further verify the role of the downstream survival pathway activated by DDP-induced ATM in chemotherapy resistance, the ATM inhibitor KU-55933 was used in combination. First, it was observed that the combination of DDP and KU-55933 increased DDP-induced apoptosis and reduced tumor cell proliferation. At the same time, KU-55933 inhibited the activation of ATM, and the expression levels of downstream p-CHK2, CtIP, and RAD51 proteins were correspondingly downregulated, while the expression of cl-PARP increased.Figure 3 ) This indicates that inhibiting ATM activation can prevent cells from performing HR repair and promote apoptosis of tumor cells. Subsequently, A549 cells were transfected with siRNA to knockdown ATM expression, further demonstrating that reducing ATM levels can also reduce the upregulation of ATM downstream proteins p-CHK2, CtIP, and RAD51 induced by DDP, thereby inhibiting HR repair and enhancing the sensitivity of A549 cells to DDP ( Figure 4 ) These results indicate that cisplatin-induced chemotherapy resistance stems from the activation of ATM in human tumor cells, which mediates the continuous activation of the downstream homologous recombination (HR) pathway.

[0125] Shikonin is a naphthoquinone component, the main chemical constituent of Lithospermum erythrorhizon, and shows good anti-tumor effects. In 2022, shikonin was identified as a broad-spectrum DNA damage response (DDR) inhibitor that can inhibit upstream DDR events by inducing the degradation of ATM and ATRIP. However, due to its hydrophobicity, low chemical stability, and high non-selective toxicity, various shikonin analogs have been successively isolated and studied. On this basis, shikonin and its six analogs were screened, and acetylakannin was found to exhibit stronger anti-tumor effects in A549 and Huh-7 cells and could significantly inhibit the activation of ATM ( Figure 6 ) Acetylakannin is a natural naphthoquinone compound that is superior to shikonin in physicochemical properties but has been less studied. For this reason, acetylakannin was used in combination with cisplatin, and it was found that acetylakannin could significantly enhance the sensitivity of tumor cells to cisplatin. The IC50 values of A549 cells and Huh-7 cells decreased from 49.54 μM to 2.2 μM and from 23.55 μM to 2.945 μM, respectively, and the sensitivities increased by 22.5-fold and 8.0-fold, respectively ( Figure 6 in A). The colony formation assay and EdU assay further confirmed that acetylakannin could restore the sensitivity of tumor cells to cisplatin ( Figure 6 in B, C). During the experiment, it was found that acetylakannin inhibited the activation of ATM and simultaneously inhibited the expression levels of HR pathway-related proteins CtIP and RAD51, thereby enhancing the sensitivity of tumor cells to cisplatin ( Figure 6 in D). More importantly, in this study, it was demonstrated for the first time that acetylakannin not only downregulates its protein level and inhibits its activation by targeting ATM, but also promotes the degradation of ATM through the Caspase pathway to exert its effect ( Figure 7 ) These findings indicate that ATM is not only a key factor for human tumor cells to develop cisplatin resistance, but also represents a therapeutic target for enhancing cisplatin sensitivity.

[0126] In summary, acetylakannin can inhibit the ATM / CHK-2 pathway, induce DNA damage in Huh-7 cells and A549 cells, and promote apoptosis. Moreover, acetylakannin can promote apoptosis and enhance the sensitivity of DDP by inhibiting the ATM pathway when combined with DDP. In addition, it was also found that acetylakannin has a strong binding affinity with ATM in A549 cells and promotes the degradation of ATM in A549 cells through a caspase-dependent pathway, thereby shortening the half-life of ATM in cells. The examples of this application illustrate the anti-tumor effect and molecular mechanism of the shikonin analogue acetylakannin, providing an experimental basis for the future clinical application of acetylakannin in the treatment of liver cancer and lung cancer.

[0127] It should be noted that this application is not limited to the above embodiments. The above embodiments are only examples, and embodiments with the same composition and the same effect within the technical scope of this application are included in the technical scope of this application. In addition, within the scope of not departing from the gist of this application, various modifications that can be conceived by those skilled in the art to the embodiments, as well as other forms constructed by combining some of the components in the embodiments, are also included in the scope of this application.

Claims

1. Use of acetylakannin or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug, characterized in that, The tumor includes liver cancer or lung cancer.

2. The application according to claim 1, characterized in that The anti-tumor drug also includes cisplatin.

3. The application according to claim 2, characterized in that, The use of acetylakannin or a pharmaceutically acceptable salt thereof as a drug resistance reverser in an anti-tumor drug.

4. The application according to claim 2, characterized in that The use of acetylakannin or a pharmaceutically acceptable salt thereof as a drug sensitizer in an anti-tumor drug.

5. The application according to claim 1, wherein The pharmaceutically acceptable salt includes a salt formed with an inorganic acid, an organic acid, an amino acid, an alkali metal or an alkaline earth metal.

6. The application according to claim 1, characterized in that The preparation raw materials of the anti-tumor drug further include pharmaceutically acceptable excipients; and / or, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a binder, a wetting agent, a lubricant, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a sweetening agent and a flavoring agent.

7. The application according to claim 1, wherein The dosage form of the anti-tumor drug includes at least one of an injection, a tablet, a patch, a suspension, a granule, a capsule, a powder, an emulsion, a solution, a pill, a dropping pill, an oral preparation, a suppository, an enema, an aerosol or a drop.

8. The application according to claim 1, wherein The use of acetylakannin or a pharmaceutically acceptable salt thereof as a cell viability inhibitor in an anti-tumor drug.

9. The application according to claim 1, characterized in that, The use of acetylakannin or a pharmaceutically acceptable salt thereof as a cell apoptosis promoter in an anti-tumor drug.

10. The application according to claim 1, characterized in that, Acetylakannin or a pharmaceutically acceptable salt thereof inhibits the expression of ATM in tumor cells.

Images