Application of sesquiterpene lactone compound in preparation of medicine for enhancing radiation therapy effect

Abstract

The invention discloses an application of a sesquiterpene lactone compound in preparation of a medicine for enhancing a radiation therapy effect, and belongs to the technical field of biomedicine.The elephantopus tomentosus monomer EM-2 in a formula (I) enhances G2 / M phase retardation caused by ionizing radiation by inhibiting a signal channel caused by the ionizing radiation, so that the sensitivity of the radiation therapy is remarkably improved. Experimental results show that the compound combined with radiotherapy can reduce the expression of a cell proliferation marker Ki67, reverse ionizing radiation induced p-AKT activation, and further enhance the anti-tumor effect of radiotherapy. The compound has a good proliferation inhibition effect on cervical cancer cells, and has low toxicity on normal human skin fibroblast HSF cells. On the other hand, EM-2 can be combined with AKT protein in a targeted mode to inhibit a PI3K / AKT signal transduction pathway activated by radiotherapy, meanwhile, protective autophagy activated after radiotherapy is inhibited, and the sensitivity of ionizing radiation to cervical cancer cells is improved.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the use of sesquiterpene lactone compounds in the preparation of drugs for enhancing the effect of radiotherapy. Background Art

[0002] Radiotherapy is currently one of the most effective cytotoxic treatments for solid tumors, and holds particular clinical value for cervical cancer. It is not only applicable across all stages of cervical cancer but is also routinely used in a variety of treatment scenarios, including postoperative adjuvant therapy for patients with high-risk factors, radical treatment for mid- to late-stage cervical cancer, and palliative care for patients in the advanced stages. Due to its precise local killing capabilities, radiotherapy holds a central position in the comprehensive treatment of cervical cancer.

[0003] Despite the remarkable efficacy of radiotherapy, tumor cell resistance to radiation remains a significant contributor to disease progression. This resistance may stem from enhanced DNA repair capacity in tumor cells, the presence of a hypoxic microenvironment, or the abnormal activation of specific signaling pathways. Radioresistance not only reduces treatment efficacy but also promotes tumor recurrence and metastasis, posing a major clinical challenge.

[0004] Furthermore, while radiotherapy kills tumor cells, it inevitably damages surrounding normal tissues, such as the intestines, bladder, and bone marrow. These side effects can lead to complications such as radiation enteritis, cystitis, or bone marrow suppression, which not only impacts patients' quality of life but may also necessitate treatment interruption or dose adjustment, thus limiting the overall efficacy of radiotherapy. Therefore, improving the selectivity of radiotherapy, enhancing tumor killing while protecting normal tissues, is a key research topic. Summary of the Invention

[0005] The present application aims to solve one of the technical problems in the above-mentioned prior art and provide a drug that can sensitize radiotherapy and ionizing radiation therapy.

[0006] The technical solution adopted by the present invention is: In a first aspect of the present application, a sesquiterpene lactone compound is provided for use in the preparation of a drug for enhancing the effect of radiotherapy, 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: (I).

[0007] More specifically, in the above technical solution, the radiotherapy is ionizing radiation.

[0008] The concentration of EM-2 is 1.25-20 μM, and the radiotherapy dose is 4-8 Gy.

[0009] More specifically, in the above technical solution, the sesquiterpene lactone compound inhibits the PI3K / AKT / mTOR signaling pathway caused by ionizing radiation.

[0010] More specifically, in the above technical solution, the sesquiterpene lactone compound enhances the G2 / M arrest caused by the ionizing radiation.

[0011] Enhancing G2 / M arrest can directly enhance radiosensitivity and significantly improve the lethality of radiotherapy. Different cell cycle stages have varying sensitivities to radiotherapy, and the biological characteristics of the arrest phase (such as DNA repair capacity and apoptosis pathways) directly influence the synergistic effect of drugs and radiotherapy. Therefore, EM-2 significantly enhances radiosensitivity by inducing G2 / M arrest in cervical cancer. However, in breast cancer, inducing different arrest phases (such as G1 or S) may result in different radiosensitization effects.

[0012] More specifically, in the above technical solution, the sesquiterpene lactone compound combined with radiotherapy reduces the level of a cell proliferation marker, and the cell proliferation marker is Ki67.

[0013] More specifically, in the above technical solution, the sesquiterpene lactone compound reverses the p-AKT activation induced by ionizing radiation.

[0014] Specifically, this reversal not only significantly enhances the sensitivity of radiotherapy but also may further improve the therapeutic effect by inhibiting the survival and repair capabilities of cancer cells. Furthermore, this effect may reduce radiotherapy resistance, prolong treatment effects, and minimize damage to normal cells, thereby reducing side effects. Ultimately, this strategy for reversing p-AKT activation provides a novel sensitization strategy for radiotherapy with important clinical application value.

[0015] More specifically, in the above technical solution, the drug is used to treat cervical cancer.

[0016] More specifically, in the above technical solution, the cervical cancer cells are Hela and SiHa.

[0017] This technical solution targets cervical cancer cells Hela and SiHa, and significantly enhances the sensitivity and efficacy of radiotherapy through the specific mechanism of action of sesquiterpenoid lactone compounds (such as inhibiting the PI3K / AKT / mTOR signaling pathway, reversing ionizing radiation-induced p-AKT activation, etc.), providing a new strategy for the treatment of cervical cancer and effectively improving the treatment effect.

[0018] Existing chemotherapy drugs are highly toxic, limiting their clinical application. However, the sesquiterpene lactone compounds in this technology selectively target cancer cells (such as HeLa and SiHa cells), potentially enhancing the effectiveness of radiotherapy while reducing toxicity to normal cells. This reduces treatment side effects and significantly improves patients' quality of life.

[0019] More specifically, in the above technical solution, the dosage form is selected from: injection, tablet, capsule, kit or patch. Those skilled in the art can prepare the above pharmaceutical composition into various dosage forms according to specific circumstances. The preparation method is a well-known technology in the art and is therefore not described in detail in this application.

[0020] In a second aspect of the present application, a sesquiterpene lactone compound is provided for use in the preparation of a sensitizer for radiotherapy, 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.

[0021] (I).

[0022] The third aspect of the present application provides the use of a sesquiterpene lactone compound in a drug for increasing cancer cell apoptosis, wherein the drug is used in radiotherapy, and the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof: (I).

[0023] In a fourth aspect of the present application, there is provided a use of a sesquiterpene lactone compound in the preparation of a drug for inhibiting cancer cell proliferation, wherein the drug is used in radiotherapy, and the sesquiterpene lactone compound is a compound represented by formula (I), a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a prodrug molecule thereof: (I).

[0024] The "pharmaceutically acceptable salt" is a conventional non-toxic salt formed by the reaction of the compound of general formula (I) with an inorganic acid or an organic acid. For example, the conventional non-toxic salt can be prepared by reacting the compound of general formula (I) with an inorganic acid or an organic acid, wherein the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid and phosphoric acid, and the organic acid includes citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid. acid, etc.; or the compound of general formula (I) forms an ester with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid, and then forms a sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt with an inorganic base; or the compound of general formula (I) forms a methylamine salt, ethylamine salt or ethanolamine salt with an organic base; or the compound of general formula (I) forms an ester with lysine, arginine or ornithine, and then forms a corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid, or a corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.

[0025] Compared with the prior art, the embodiments of the present application have the following beneficial effects: 1. The inventors discovered that the sesquiterpene lactone compound can effectively and specifically intervene in the PI3K / AKT / mTOR pathway. Blocking this pathway with a specific inhibitor significantly reduced cell survival, confirming that abnormal activation of the PI3K / AKT / mTOR pathway is a key molecular mechanism mediating radioresistance in cervical cancer. Further studies have shown that compound EM-2 can effectively inhibit PI3K / AKT / mTOR pathway signaling by directly targeting the kinase domain of the AKT protein.

[0026] 2. The sesquiterpene lactone compounds can enhance radiotherapy sensitivity by inducing autophagic flux inhibition. Radiotherapy is accompanied by changes in typical autophagy markers, such as an increase in the LC3-II / LC3-I ratio and upregulated Beclin-1 expression. When combined with radiotherapy, autophagy inhibitors significantly reduced cell survival, suggesting that blocking autophagy activation can effectively reverse tumor cell radioresistance. EM-2 effectively inhibits radiotherapy-induced autophagic flux, a phenomenon closely related to its radiosensitizing effect.

[0027] 3. EM-2 and radiotherapy exhibit significant synergistic effects, potentially leading to clinical translational advantages. In vitro experiments demonstrated that the combination of EM-2 and radiotherapy produced a significant synergistic antitumor effect (synergy index CI < 0.9). In an in vivo nude mouse cervical cancer xenograft model, the EM-2 combined with radiotherapy group exhibited a higher tumor growth inhibition rate than the radiotherapy alone group. Pathological examination revealed no significant toxic damage to major organs. These results suggest that EM-2, as a novel radiosensitizer, possesses significant potential for clinical translational applications.

[0028] 4. Enhancing G2 / M arrest can directly enhance radiosensitivity and significantly improve the lethality of radiotherapy. Different cell cycle stages have varying sensitivities to radiotherapy, and the biological characteristics of the arrest phase (such as DNA repair capacity and apoptosis pathways) directly influence the synergistic effect of drugs and radiotherapy. Therefore, EM-2 significantly enhances radiosensitivity by inducing G2 / M arrest in cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 Schematic diagram of the PI3K / AKT / mTOR signaling pathway activated by radiotherapy in cervical cancer cells.

[0031] Figure 2 Schematic diagram of how inhibiting autophagy enhances the radiosensitivity of cervical cancer cells.

[0032] Figure 3 Schematic diagram of the synergistic effect of EM-2 and radiotherapy against cervical cancer.

[0033] Figure 4 Schematic diagram of EM-2 targeting and binding to AKT protein.

[0034] Figure 5 Schematic diagram of how EM-2 enhances radiosensitivity by inhibiting the PI3K / AKT signaling pathway.

[0035] Figure 6 Schematic diagram of how EM-2 enhances radiosensitivity by inhibiting autophagy.

[0036] Figure 7 Schematic diagram of EM-2 enhancing the anti-tumor efficacy of radiotherapy in a nude mouse cervical cancer xenograft model.

[0037] Figure 8Schematic diagram of how EM-2 enhances radiosensitivity in vivo by dually inhibiting the PI3K / AKT signaling pathway and autophagy. DETAILED DESCRIPTION

[0038] In order to more clearly understand the technical content of the present invention, the following examples are specifically described in detail in conjunction with the accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0039] EM-2 is a sesquiterpene lactone compound extracted from the traditional Chinese herbal medicine Euphorbia pulegium, named 2β-methoxy-2-deethoxyphantomolin, with a molecular formula of C 20 H 24 O6, with a molecular weight of 360, was isolated and purified by the School of Pharmacy of Jinan University. Its structure is shown below (I). EM-2 powder was dissolved in DMSO to a 40 mM mother liquor, aliquoted, and stored at -80°C.

[0040] (I).

[0041] 1. Materials and Methods Cell lines: Four cervical cancer cells, Hela cells, SiHa cells, Caski cells, and C33A cells, and normal human skin fibroblasts (HSF) were purchased from the American type culture collection (ATCC).

[0042] Table 1 Antibodies required for the experiment and their suppliers: Antibody name company Anti-β-actin Cell Signaling Technology Anti-PI3K Cell Signaling Technology Anti-Phospho-PI3K (Ser249) Cell Signaling Technology Anti-AKT Cell Signaling Technology Anti-Phospho-AKT (Ser473) Cell Signaling Technology Anti-Phospho-AKT (Thr308) Cell Signaling Technology Anti-mTOR Cell Signaling Technology Anti-Phospho-mTOR (Ser2448) Cell Signaling Technology Anti-Beclin1 Cell Signaling Technology Anti-P62 Cell Signaling Technology Anti-LC3B Cell Signaling Technology Goat anti-rabbit IgG-HRP Proteintech Goat anti-mouse IgG-HRP Proteintech Methods: This study evaluated the inhibitory effect of EM-2 on the proliferation of cervical cancer cells and its synergistic effect with radiotherapy by CCK-8 assay, and calculated the combination index (CI) to analyze drug interactions. The effect of EM-2 on the survival of cancer cells after radiotherapy was detected by clonogenic assay. Flow cytometry was used to analyze the effects of EM-2 combined with radiotherapy on cell apoptosis and cell cycle distribution. Molecular docking technology was used to predict the binding pattern of EM-2 and AKT protein. Western blot and qRT-PCR were used to investigate the regulation of EM-2 on the PI3K / AKT / mTOR signaling pathway and the expression of autophagy-related proteins. Transmission electron microscopy was used to observe the effect of radiotherapy on autophagosome formation. Finally, a nude mouse xenograft tumor model was used to verify the in vivo radiosensitization effect of EM-2 and its safety assessment.

[0043] CCK8 experiment: (1) Take cells in the logarithmic growth phase and digest them with trypsin. After about 1-3 minutes, add culture medium containing 10% FBS to terminate the digestion. Centrifuge at 800 rpm for 3 minutes at room temperature, remove the supernatant, and resuspend the cells in culture medium containing 10% FBS to make a single cell suspension and count them. Take a 96-well plate and add 100 μL of cell suspension at a density of 3000 cells per well. Set up 3-5 replicate wells for each group. Fill the outer circle of the well plate with 200 μL of 1× PBS to reduce evaporation of the culture medium. Place in an incubator for culture.

[0044] (2) Using the serial dilution method, the drug was diluted to twice the experimental concentration. 100 μL of drug was added to the cells to dilute the drug concentration to twice the experimental working concentration. At the same time, different doses of radiotherapy (IR) were treated. After 72 h of culture, 100 μL of fresh culture medium containing 10% CCK8 solution was added to each well of the zero adjustment group, control group, and experimental group, and then cultured for another 1 h. The absorbance at 450 nm was measured using a multifunctional microplate reader, and the cell survival rate of each group was calculated by calculating the average absorbance value of each group.

[0045] (3) Cell viability (%) = (average absorbance of the experimental group - average absorbance of the zero-adjustment group) / (average absorbance of the control group - average absorbance of the zero-adjustment group) × 100%. GraphPad Prism was used to calculate the proliferation inhibition curve of cervical cancer cells.

[0046] CI (Combination index) value calculation: (1) Take cells in the logarithmic growth phase and digest them with trypsin. After about 1-3 minutes, add culture medium containing 10% FBS to terminate the digestion. Centrifuge at 800 rpm for 3 minutes at room temperature, remove the supernatant, and resuspend the cells in culture medium containing 10% FBS to make a single cell suspension and count them. Take a 96-well plate and add 100 μL of cell suspension at a density of 3000 cells per well. Set up 3-5 replicate wells for each group. Fill the outer circle of the well plate with 200 μL of 1× PBS to reduce evaporation of the culture medium. Place in an incubator for culture.

[0047] (2) When the cells reach the logarithmic growth phase, trypsinize the cells with 0.25% EDTA-containing trypsin solution. Terminate the digestion with culture medium containing 10% FBS and pipette into a single-cell suspension. Collect the cells by low-speed centrifugation at 800 rpm for 3 min. Resuspend the cells in culture medium and calculate the cell density. At a density of 4000-6000 cells per well, seed 100 μL of the cell suspension into a 96-well plate and continue to culture overnight. Fill the periphery of the well plate with 200 μL of 1× PBS to reduce evaporation of the culture medium.

[0048] (3) EM-2 was applied to Hela cells and SiHa cells alone or in combination at concentrations of 1.25, 2.5, 5, 10, and 20 μM and a radiotherapy dose of 4 Gy or 8 Gy. After 72 h of culture, 100 μL of fresh culture medium containing 10% CCK8 solution was added to each well of the zero adjustment group, control group, and experimental group, and the cells were cultured for another 1 h. The absorbance at 450 nm was measured using a multifunctional microplate reader, and the cell survival rate of each group was calculated by calculating the average absorbance value of each group.

[0049] (4) Calculate cell survival rate and calculate the combination index (CI) value using CompuSyn software. CI < 0.9 indicates synergistic effect; 0.9 < CI < 1.1 indicates additive effect; CI > 1.1 indicates antagonistic effect.

[0050] Colony formation assay: (1) Take cells in the logarithmic growth phase and digest them with trypsin solution containing 0.25% EDTA. After low-speed centrifugation, remove the supernatant and resuspend in culture medium containing 10% FBS. After counting the cells, evenly seed the cells in a six-well plate with approximately 500-1000 cells per well. Gently shake the six-well plate and observe under a microscope to ensure that the cells are evenly spread in the six-well plate. Then place the plate in a cell culture incubator.

[0051] (2) After the cells have attached, remove the supernatant and, according to the experimental protocol, add 2 mL of culture medium containing different concentrations of drugs to each well to treat the cells while simultaneously receiving radiotherapy. The six-well plate is then placed in a cell culture incubator and incubated for 7 to 14 days.

[0052] (3) After the culture is complete, discard the supernatant. Slowly add 1 mL of 1× PBS to the wells along the wall of the 6-well plate and gently shake to wash away the remaining supernatant. Be careful not to blow the cells with a pipette to prevent the adherent cells from being washed off. Repeat this washing process twice. Then, add 1 mL of 4% paraformaldehyde solution to each well to fix the cells for 30 minutes to 1 hour.

[0053] (4) After the treatment time, remove the culture medium, gently rinse with 1× PBS 2-3 times, add 1 mL of 4% paraformaldehyde solution, and fix the cells for 20 minutes. Discard the fixative, gently shake and wash with 1× PBS 1-2 times, then add 1 mL of 0.1% crystal violet staining solution and stain the cells for 30 minutes. Aspirate the 0.1% crystal violet staining solution, add tap water to rinse 2-3 times, invert and dry, and take pictures under a film observation light to record the colony formation. A colony of cells with more than 50 cells in close proximity is counted as one clone. Statistical processing is performed using ImageJ software.

[0054] Flow cytometry cycle analysis: (1) Set up a control group and an experimental group. Take cells in the logarithmic growth phase and evenly seed them into a six-well plate at a density of approximately 200,000 cells per well. Place them in a cell culture incubator overnight to allow the cells to adhere to the wall. After the cells adhere, treat the cells (radiotherapy) according to the experimental requirements.

[0055] (1) After 24 or 48 hours, remove the 6-well plate and collect the culture medium in the wells into a centrifuge tube. Wash each well twice with 1 mL of 1× PBS, and aspirate the PBS into the centrifuge tube. Add an appropriate amount of trypsin to the wells to digest the cells. Transfer the wells to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Be careful to operate gently to avoid physical damage to the cells.

[0056] (2) Discard the supernatant, add 1 mL of PBS to resuspend the cells, repeat the centrifugation and discard the supernatant. Add 2 mL of pre-chilled 70% ethanol to each tube and transfer to a 4°C refrigerator to fix overnight.

[0057] (3) The next day, the cells were centrifuged again at 1000 rpm for 5 min at room temperature. The supernatant was removed and the cells were resuspended in pre-chilled PBS. The cells were centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was removed and the PI solution was prepared. 25 μL of PI staining solution and 10 μL of RNase A were added to every 0.5 mL of staining buffer. 1 mL of staining buffer was added to each sample, and the cells were thoroughly mixed. The cells were incubated at 37°C in the dark for 30 min and then stored in a refrigerator at 4°C. The cell cycle distribution of each group was detected by flow cytometry, and the DNA content of each group was analyzed using ModFit software.

[0058] Annexin V-FITC / PI staining and flow cytometry experiments: (1) Set up a control group and an experimental group. Take cells in the logarithmic growth phase and evenly seed them into a six-well plate at a density of approximately 200,000 cells per well. Place them in a cell culture incubator overnight to allow the cells to adhere to the wall. After the cells adhere, treat the cells (radiotherapy) according to the experimental requirements.

[0059] (2) After 24 or 48 hours, remove the 6-well plate and collect the culture medium in the wells into a centrifuge tube. Wash each well twice with 1 mL of 1× PBS, and aspirate the PBS into the centrifuge tube. Add an appropriate amount of trypsin to the wells to digest the cells. Transfer the wells to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Be careful to operate gently to avoid physical damage to the cells.

[0060] (3) Discard the supernatant, add 1 mL of PBS to resuspend the cells, repeat centrifugation and discard the supernatant, retaining the cell pellet. Use the Annexin V / PI double staining cell apoptosis kit, add Binding Buffer, Annexin V, and PI staining solution in a 100:1:1 ratio by volume, suspend and mix the cells, protect from light, incubate at room temperature for 10 minutes, and use an intelligent analytical flow cytometer to analyze the cell apoptosis and draw a graph.

[0061] Molecular docking: (1) The AKT protein sequence was obtained from the Uniprot database, the crystal structure was obtained by homology modeling using Swissmodel, and the small molecule 3D structure was obtained from the Pubchem database (https: / / pubchem.ncbi.nlm.nih.gov / ).

[0062] (2) Connect to the CB-DOCK2 online server ( https: / / cadd.labshare.cn / Blind docking is completed using CB-DOCK2 (cb-dock2 / php / blinddock.php). CB-DOCK2 uses an artificial neural network for cavity detection and Autodock Vina for docking.

[0063] (3) The docked complexes were selected based on binding energy, and the interaction forces and docking positions were analyzed to evaluate the binding stability. The interaction force analysis between the ligand and receptor was performed using the PLIP online service (https: / / pliptool.biotec.tu-dresden.de / plip-web), and the 3D conformation of the ligand-receptor complex was displayed using Pymol software.

[0064] Western blot experiment: (1) Protein extraction. Cells in the logarithmic growth phase were seeded in 6-well plates and grouped. After overnight adherent culture, radiotherapy was performed. After 24-48 hours, the supernatant culture medium was aspirated and the excess culture medium was washed with PBS. The cells were collected and lysed by adding RIPA strong lysis buffer containing 1% protease inhibitors and 1% protein phosphatase inhibitors. Lysis was carried out on ice for 30 minutes. The lysed cells were then transferred to a centrifuge tube and centrifuged in a pre-cooled high-speed centrifuge (4°C, 12000 rpm) for 15 minutes to obtain the protein supernatant. The protein concentration of the protein supernatant was determined using a BCA micro-protein concentration assay kit. Then, 1 / 4 volume of 5× SDS-PAGE protein loading buffer was added, mixed, and heated in a metal bath at 100°C for 5 minutes to denature the protein. The obtained protein sample was stored at -80°C.

[0065] (2) Gel electrophoresis and transfer. Assemble the 4-20% protein precast gel with the electrophoresis device, add 1× electrophoresis solution to the inner and outer tanks, then load the rainbow protein Maker and protein sample together for electrophoresis. Run the electrophoresis (120 V) for about 30 minutes until the indicator band approaches the bottom of the precast gel. Stop the electrophoresis, remove the gel, cut the gel where the target protein is located according to the Maker band, and place it flat on a 0.45 or 0.22 μM PVDF membrane (select membranes of different thicknesses according to the molecular weight of the target protein, and soak the membrane in methanol for activation in advance). After confirming that the membrane and the gel are completely attached, insert the transfer clip and place it in the transfer tank. Fill it with 1× transfer solution and transfer the membrane at a constant current of 250 mA for 90 minutes. Fill the periphery of the transfer tank with ice to cool it down.

[0066] (3) Blocking and antibody incubation. After transfer, soak the membrane in 5% blocking milk and incubate on a shaker at room temperature for 1 h. Then, wash off the excess milk with 1× TBST buffer and soak the membrane in the corresponding primary antibody dilution solution and incubate overnight (4°C). After the primary antibody incubation, wash the membrane three times with 1× TBST buffer. Then, place the membrane in the corresponding secondary antibody dilution solution and incubate on a shaker at room temperature for 1.5 h. After the end, wash the membrane three times with 1× TBST buffer.

[0067] (4) Luminescent development of protein bands. After washing the membrane, apply ultrasensitive ECL chemiluminescent solution to the membrane, expose it using a fully automatic chemiluminescence image analysis system, collect images, and analyze the resulting bands.

[0068] qRT-PCR experiments: (1) Total RNA extraction and sample lysis: Hela cells and SiHa cells in good condition were taken and digested into cell suspension according to the routine method. The density was adjusted to 1 × 10 5 Cells were seeded at 1 μM / mL in a 6-well plate and allowed to adhere overnight in a 37°C incubator. EM-2 (0, 0.25, 0.5, and 1 μM) was then added. For combination experiments, a blank control group (no radiotherapy, no drug) was established, EM-2 alone was administered at a concentration of 1 μM, the radiotherapy group received a dose of 2 Gy, and the combination group received EM-2 (1 μM) plus radiotherapy (2 Gy). Twenty-four hours after radiotherapy, the culture medium was removed, the cells were rinsed twice with PBS, and the adherent cells were trypsinized to a cell suspension in a 1.5 mL centrifuge tube. The suspension was centrifuged at 500 rpm for 3 minutes at room temperature. The supernatant was aspirated, and 500 μL of lysis buffer was added to each tube. The cells were vigorously pipetted at least 10 times and vortexed to mix thoroughly. The remaining RNA binding, column washing, and RNA elution procedures were the same as above.

[0069] (2) Removal of genomic DNA: Prepare the following system on ice and incubate at 42°C for 2 min.

[0070] (3) Reverse transcription reaction: Mix 20 μL of the system, incubate at 37°C for 15 min, incubate at 85°C for 5 sec, and store at 4°C.

[0071] (4) qPCR System configuration (cDNA template is the product of reverse transcription reaction, diluted tenfold before use): qPCR procedure: Pre-denaturation: 95°C for 30 seconds; PCR reaction: Denaturation: 95°C for 5s, annealing: 60°C for 30-60s, extension: 72°C for 30-60s; Melting curve: 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds; Repeat 40 times.

[0072] The primer sequences are shown in Table 2.

[0073] Table 2 qPCR primer sequences Transmission electron microscopy cell observation: (1) Cervical cancer Hela and SiHa cells were collected, the cell pellets were resuspended in pre-cooled electron microscopy fixative, and fixed in a 4°C refrigerator for 2 h.

[0074] (2) After resuspending in pre-cooled PBS, fix again with 1% osmium hydroxide solution in a 4°C refrigerator for 1 h.

[0075] (3) Dehydrate in a gradient of ethanol (50%, 70%, 90%, 100%) for 10 minutes per step. Replace the ethanol with acetone and dehydrate further. Place the sample in a mixture of epoxy resin and acetone (1:1) and infiltrate at room temperature for 2 hours. Replace with pure epoxy resin and infiltrate overnight at room temperature.

[0076] (4) Place the sample in a mold, add fresh epoxy resin, and polymerize at 60°C for two days. Use an ultramicrotome to cut the embedded block into 50-70 nm slices.

[0077] (5) The embedded sections were collected on copper grids and double-stained with uranyl acetate and lead citrate for 5 min each. They were then observed using a HITACHI transmission electron microscope.

[0078] Immunofluorescence experiments: (1) Take Hela and SiHa cells in good condition and prepare a cell suspension by routine digestion. Prepare a 24-well plate and place a 14 mm sterile round sterile slide in the plate in advance. Wet the bottom of the plate with culture medium in advance. Adjust the density to 10,000 cells / well and inoculate them in the 24-well plate. Culture in a 37°C incubator overnight.

[0079] (2) After the cells adhered, drugs of corresponding concentrations were added to each well, and the cells were treated with radiotherapy and cultured for another 24 h.

[0080] (3) After removing the culture medium, wash the cells twice with pre-cooled 1× PBS, add 100 μL of 4% paraformaldehyde, and fix the cells for 20 min.

[0081] (4) Aspirate the 4% paraformaldehyde, wash twice with pre-cooled 1× PBS, then add 0.1% Triton-X and incubate at room temperature for 10 min.

[0082] (5) Add 10% goat serum to just cover the cells and block at 37°C for 1 h.

[0083] (6) After removing the 10% goat serum and washing with pre-cooled 1× PBS, the primary antibody was diluted with 5% BSA at a ratio of 1:200 to make a working solution, incubated at 4°C overnight, and then rinsed with 1× PBS containing 2% Tween 20 and washed twice.

[0084] (7) Add the corresponding fluorescent secondary antibody until just covering the cells at a dilution ratio of 1:100 and incubate at room temperature for 1 h. Aspirate the secondary antibody and wash twice with 1× PBS containing 2% Tween 20.

[0085] (8) Add 100 μL of ready-to-use DAPI staining solution to each well and incubate at room temperature in the dark for 10 min.

[0086] (9) Use a paper towel to absorb the liquid on the slide, carefully remove the slide, add a drop of anti-fluorescence quenching sealing liquid on the slide, place the slide upside down on the slide, and fix the slide with armor oil to prevent it from moving, and then observe the fluorescence expression under an inverted fluorescence microscope.

[0087] Plasmid transfection: (1) Take cells in logarithmic growth phase, 8 × 10 5 The cells in each well were seeded into a 6-well plate at a density of 100 μg / mL, cultured with complete culture medium without double antibodies, and placed in an incubator to continue culture overnight to adhere to the wall.

[0088] (2) Dilute the plasmid solution and the transfection reagent according to the instructions of the transfection reagent. Gently mix the diluted plasmid solution and the transfection reagent and let it stand at room temperature for 15 minutes.

[0089] (3) Aspirate the complete culture medium without the double antibody from the cell culture and rinse the cells twice with PBS.

[0090] (4) Mix the transfection complex with complete culture medium without double antibody and add it to the well plate. Gently shake the 6-well plate to evenly distribute the mixture. Simultaneously, the treatment group is treated with drugs according to the experimental protocol.

[0091] (5) Continue culturing in a CO2 incubator for 24 h before conducting subsequent experiments.

[0092] Animal experiments: (1) Tumor cells were injected subcutaneously to establish a nude mouse xenograft tumor model. BALB / c-nu nude mice were used, and 800 × 10 HeLa cells were inoculated subcutaneously in each mouse. 4 When the tumor grows to 100 mm 3 When the number is about , group processing is performed.

[0093] (2) The mice were divided into 1. saline group; 2. EM-2 group (3 mg / kg); 3. radiotherapy group (10 Gy); 4. EM-2 (3 mg / kg) + radiotherapy (IR) (10 Gy) group. The drug group received intraperitoneal injection of drug (EM-2) once every other day, with each dose of 3 mg / kg, for a total of 7 times. The radiotherapy group avoided important organs of the mice, with each radiotherapy dose of 2 Gy, for a total of 5 times. During the experiment, the tumor growth was observed, the tumor volume was measured every 2 days, the longest diameter a and the shortest diameter b of the tumor were measured, and the a*b 2 Tumor volume V was calculated using the formula [number missing] / 2, and tumor growth curves were plotted. On day 15, mice were euthanized (anesthetized and cervical dislocation) in accordance with animal ethics. Tumor tissue was removed and weighed. Hearts, livers, kidneys, spleens, and other organs from a subset of mice in each group were collected for HE staining and photographed.

[0094] Data statistics and analysis The experiments described above were repeated three times. One representative experiment from three independent experiments is shown. Data are presented as mean ± standard deviation (mean ± SD). Two-tailed T-tests were used between two groups, and ANOVA analysis of variance was used for comparisons of more than two groups. P < 0.05 was considered significant. All statistical analyses were performed using SPSS 26. Images were processed using Adobe Illustrator 2021.

[0095] Example 1 Radiotherapy is an important cancer treatment. To investigate the specific survival pathways activated by cancer cells to induce radioresistance, the inventors used the CCK-8 assay to examine the effects of different doses of radiotherapy (IR) on the proliferation of cervical cancer Hela, SiHa, C33A, and CaSki cells. All four cell lines showed dose-dependent growth inhibition, but the cytotoxic effect of IR was not significant. Even at 8 Gy, the inhibition rate was less than 50%. Figure 1 A). We selected Hela cells, which are the least sensitive to radiotherapy, for RNA sequencing, and the results suggested that the PI3K / AKT / mTOR pathway is a key pathway for cervical cancer radiation resistance ( Figure 1 B). GSEA analysis provides further support for the activation of the PI3K / AKT / mTOR pathway in radiotherapy-treated cervical cancer cells ( Figure 1 C). Western blot confirmed that in Hela and SiHa cells, the expression of key phosphorylated proteins in the radiotherapy-induced PI3K / AKT / mTOR pathway was upregulated with increasing radiotherapy dose ( Figure 1 D). Quantitative analysis of grayscale intensity of Western blot bands confirmed the activation of the PI3K / AKT / mTOR pathway ( Figure 1 E). The above results demonstrate that radiotherapy activates the expression of PI3K / AKT / mTOR pathway in cervical cancer cells.

[0096] Example 2 Autophagy plays a role in both survival and death of cancer cells. Transmission electron microscopy (TEM) shows characteristic ultrastructural changes in irradiated cells, including mitochondrial damage (swelling and cristae fragmentation) and an increase in autophagolysosomes with a typical fragmented morphology ( Figure 2 A). The inventors showed through colony formation experiments that chloroquine (CQ) significantly enhanced the radiotherapy-mediated inhibition of colony formation ( Figure 2 B, C), indicating that autophagy can serve as a cellular protection mechanism during radiotherapy. To dynamically monitor autophagic flux, the inventors used confocal microscopy to observe the GFP-mCherry-LC3 dual fluorescence system. CQ prevented GFP fluorescence degradation, while rapamycin (RAPA), an mTOR inhibitor that activates autophagy, promoted GFP quenching. Analysis of GFP and mCherry fluorescence signals revealed that radiotherapy significantly reduced GFP intensity, accompanied by the accumulation of autophagosomes in the cytoplasm, indicating that autophagic flux in cells was enhanced after radiotherapy ( Figure 2 D). In summary, radiotherapy activates autophagy in cervical cancer cells, and inhibiting autophagy can sensitize these cells to radiotherapy.

[0097] Example 3 The inventors further investigated the potential of EM-2 as a radiosensitizer for cervical cancer. The inhibitory effect of EM-2 on the proliferation of cervical cancer cells was examined. We also evaluated the cytotoxicity of EM-2 on normal human skin fibroblast (HSF) cells ( Figure 3 A). The IC50 value of EM-2 for HSF cells was higher than that for cervical cancer cells, indicating that EM-2 has good selectivity for cancer cells. Compared with radiotherapy alone, the combination of EM-2 and radiotherapy (4 Gy or 8 Gy) significantly reduced cell viability, with a combination index (CI) value below 0.9, confirming a strong synergistic effect ( Figure 3 BE). Isobologram analysis visually confirmed the synergistic effect between EM-2 and radiotherapy ( Figure 3 F). The combined treatment also induced a significantly increased apoptosis rate ( Figure 3 G). Cell cycle analysis showed that EM-2 combined with radiotherapy significantly changed the cell cycle distribution of Hela and SiHa cells ( Figure 3 H), radiotherapy alone caused a partial arrest in the G2 / M phase, while radiotherapy combined with EM-2 significantly enhanced the G2 / M phase arrest induced by radiotherapy, suggesting that EM-2 increases the sensitivity of cervical cancer cells to radiotherapy by promoting cell cycle arrest. Clonogenic analysis further demonstrated that EM-2 combined with radiotherapy significantly inhibited colony formation ( Figure 3 I). Taken together, these results suggest that EM-2 and radiotherapy synergistically inhibit cervical cancer cells.

[0098] Example 4 Molecular docking showed that EM-2 and AKT proteins were stably bound through hydrophobic interactions and hydrogen bonds ( Figure 4 ).

[0099] Example 5 In Hela and SiHa cells, EM-2 treatment reduced AKT phosphorylation in a dose-dependent manner, as confirmed by Western blot and EM-2 quantitative analysis ( Figure 5 A, B). A complementation experiment was performed using AKT-overexpressing (OE-AKT) cells to verify the inhibitory effect of EM-2 on AKT. Western blot showed that treatment partially reversed the increase in AKT phosphorylation induced by OE-AKT, indicating that EM-2 can inhibit AKT activity ( Figure 5 C, D). To determine whether EM-2 can inhibit radiation-induced activation of the PI3K / AKT / mTOR pathway, we performed Western blot experiments. Compared with radiotherapy alone, EM-2 combined with radiotherapy significantly inhibited radiotherapy-induced activation of the PI3K / AKT / mTOR pathway and reduced the levels of p-PI3K and p-AKT ( Figure 5E, F). EM-2 also reduced AKT mRNA levels and partially reversed radiotherapy-induced AKT transcriptional activation ( Figure 5 G). Immunofluorescence confirmed that EM-2 combined with radiotherapy significantly reduced the level of p-AKT ( Figure 5 H). These results suggest that EM-2 enhances radiosensitivity by inhibiting PI3K / AKT / mTOR activation, supporting its synergistic role in the treatment of cervical cancer.

[0100] Example 6 Western blot analysis showed that EM-2 dose-dependently decreased the expression of Beclin1 and increased the expression of P62 in Hela and SiHa cells, indicating that autophagy was inhibited ( Figure 6 A, B). The autophagy inducer rapamycin (RAPA) complementation experiment showed that EM-2 partially reversed RAPA-induced autophagy activation, reduced the expression of Beclin1 and LC3B-II, and increased the level of P62 ( Figure 6 C, D). Similarly, EM-2 inhibited radiotherapy-induced autophagy activation, as demonstrated by decreased Beclin1 and LC3B expressions and increased P62 levels ( Figure 6 E, F). LC3B is a key marker of autophagosome formation, reflecting autophagic activity. EM-2 also dose-dependently reduced the mRNA level of MAP1LC3B and partially reversed radiotherapy-induced MAP1LC3B transcription ( Figure 6 G). Immunofluorescence confirmed that EM-2 combined with radiotherapy significantly reduced the fluorescence intensity of LC3B compared with radiotherapy alone ( Figure 6 H). Taken together, these findings suggest that EM-2 enhances radiosensitivity by inhibiting autophagy.

[0101] Example 7 To verify the in vivo effect of EM-2 combined with radiotherapy, we used a nude mouse cervical cancer xenograft model ( Figure 7 A). Photo of tumor ( Figure 7 B) shows that the combination of EM-2 and radiotherapy is more effective than either treatment alone. Tumor volume was monitored throughout the treatment period ( Figure 7 C). The tumors in the control group grew rapidly, with an average tumor volume of (1051.6±229.4) mm3 on day 13. The combined radiotherapy (IR) + EM-2 group had the highest tumor inhibition rate, with a tumor volume of (278.6±81.3) mm3 on day 13, a 73.5% reduction compared to the control group. At the end of the experiment, the tumors were resected and weighed ( Figure 7D). The average tumor weight in the control group was (1.14±0.27) g, while the average tumor weights in the radiotherapy group and the EM-2 monotherapy group were (0.69±0.14) g (inhibition rate 39.4%) and (0.61±0.18) g (inhibition rate 46.5%), respectively. The combination group had the lowest tumor weight, at (0.37±0.16) g, with a tumor inhibition rate of 67.5%. There were no significant differences in body weight among the groups ( Figure 7 E), no abnormal changes in food intake or activity were observed during treatment. To evaluate the safety of EM-2 combined with radiotherapy, we performed serum biochemical indices ( Figure 7 F) and histopathological analysis of major organs (heart, liver, spleen, lung, kidney) ( Figure 7 G). Serum ALT, AST, UA, and CREA levels, which reflect liver and kidney function, showed no statistically significant differences between the groups. Histopathological analysis of major organs (heart, liver, spleen, lungs, and kidneys) revealed no abnormalities, confirming the safety of the combined therapy. In summary, EM-2 combined with radiotherapy demonstrated significant antitumor efficacy without causing toxicity to major organs, demonstrating the potential of EM-2 as a safe and effective treatment strategy for cervical cancer.

[0102] Example 8 To evaluate the histopathological effects of EM-2 combined with radiotherapy on transplanted tumors, H&E staining was performed on the transplanted tumors. It was found that EM-2 combined with radiotherapy significantly reduced tumor cell density, nuclear staining intensity, and nuclear division figures, and extensive necrosis and fibrotic stroma were observed ( Figure 8 A). Immunohistochemical analysis showed that the combined treatment significantly reduced the expression of Ki67 and p-AKT, while increasing the level of P62, indicating that EM-2 inhibited tumor proliferation, suppressed AKT signaling, and reversed radiotherapy-induced autophagy ( Figure 8 B, C). Western blot experiments of AKT, p-AKT, Beclin1, P62, and LC3B in tumor tissues confirmed these findings ( Figure 8 D) EM-2 monotherapy reduced p-AKT levels, whereas radiotherapy increased p-AKT levels. Combination therapy reversed radiotherapy-induced p-AKT activation, consistent with the IHC results. Similarly, radiotherapy alone induced autophagy, with increased Beclin1 and LC3B expression and decreased p62 expression. Combination treatment reversed these effects, confirming the ability of EM-2 to inhibit radiation-induced autophagy. In summary, EM-2 enhances the antitumor effect of radiotherapy in vivo by dually inhibiting the PI3K / AKT / mTOR signaling pathway and autophagy, highlighting the potential of EM-2 as a synergistic therapeutic strategy with radiotherapy.

[0103] In summary, this study found that EM-2 enhances radiosensitivity in cervical cancer through a dual mechanism: firstly, by directly targeting the AKT protein, inhibiting activation of the PI3K / AKT / mTOR signaling pathway, and secondly, by blocking the protective autophagy process. This dual inhibitory effect collectively enhances the killing effect of radiotherapy on cervical cancer cells. This study, for the first time, elucidates the mechanism of action of the novel sesquiterpene lactone compound EM-2 as a radiosensitizer, providing important experimental evidence for the development of EM-2-based radiosensitization treatments for cervical cancer and holds significant promise for clinical application.

[0104] Research has discovered that a sesquiterpene lactone compound (EM-2) can enhance the sensitivity of cervical cancer cells to radiotherapy through a dual mechanism. On the one hand, EM-2 reverses radiotherapy-induced p-AKT activation by inhibiting the PI3K / AKT / mTOR signaling pathway, significantly enhancing the antitumor effect of radiotherapy. On the other hand, EM-2 further impairs cancer cell proliferation and survival by arresting cells in the G2 / M phase. Furthermore, EM-2 enhances the cytotoxic effects of radiotherapy by inhibiting the radiotherapy-activated autophagy pathway. Experimental results demonstrated that EM-2 combined with radiotherapy exhibited significant synergistic effects in HeLa and SiHa cervical cancer cells, significantly reducing the expression of the cell proliferation marker Ki67 and significantly inhibiting tumor growth and metastasis, with good selectivity over normal cells. In vivo experiments further validated the significant antitumor efficacy and favorable safety profile of EM-2 combined with radiotherapy, providing a novel radiosensitization strategy for cervical cancer with important clinical application value.

[0105] The above-described embodiments merely represent several embodiments of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person of ordinary skill in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. Use of a sesquiterpene lactone compound in the preparation of a drug for enhancing the effect of radiotherapy, 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: （I）。 2. The use according to claim 1, characterized in that The radiation therapy is ionizing radiation.

3. The use according to claim 2, characterized in that The sesquiterpene lactone compound inhibits the PI3K / AKT / mTOR signaling pathway caused by ionizing radiation.

4. The use according to claim 3, characterized in that The sesquiterpene lactone compound enhances the G2 / M phase arrest caused by ionizing radiation.

5. The use according to claim 4, characterized in that The sesquiterpene lactone compound combined with radiotherapy reduces the expression of a cell proliferation marker, which is Ki67.

6. The use according to claim 5, characterized in that The sesquiterpene lactone compound reverses the p-AKT activation induced by ionizing radiation.

7. The use according to any one of claims 1 to 6, characterized in that The medicine is used to treat cervical cancer.

8. The use according to claim 7, characterized in that The cervical cancer cells are Hela and SiHa.

9. The use according to any one of claims 1 to 6, characterized in that The dosage form is selected from the group consisting of injection, tablet, capsule, kit or patch.

10. Use of a sesquiterpene lactone compound in the preparation of a sensitizer for radiotherapy, 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: （I）。

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