Application of combination of dihydroartemisinin and chrysin in preparation of product for improving sensitivity of tumor cells to TOP2A inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of dihydroartemisinin combined with chrysin in preparation of a product for improving sensitivity of tumor cells to a TOP2A inhibitor. Wherein the TOP2A inhibitor is etoposide and is specifically used for hindering DNA repair. The invention provides a new solution for the drug resistance of the existing TOP2A inhibitor, fully utilizes and prolongs the life cycle of the existing drug, and is more in line with the current medical industry level and the current medical insurance investment situation in the principle of medical economics.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of dihydroartemisinin combined with chrysin in the preparation of a product that improves the sensitivity of tumor cells to TOP2A inhibitors. Background Art

[0002] Lung cancer is one of the common malignant tumors that threaten human life, with extremely high morbidity and mortality. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The main treatments for lung cancer include surgery, radiotherapy, chemotherapy, immunotherapy, and targeted drug therapy. However, the chemotherapy drugs currently widely used in clinical practice still have serious toxic side effects, such as nausea and vomiting, bone marrow transplantation, cardiotoxicity, neurotoxicity, etc. These side effects may worsen the patient's condition. Similarly, the problems to be solved in cancer include tumor heterogeneity and drug resistance. Exploring low-toxic and effective new anticancer drugs is the focus of attention of scientists at home and abroad. Studies have found that the combination of different drugs can partially improve the above problems.

[0003] Etoposide (VP-16), with the chemical formula C29H32O13, is an effective topoisomerase II inhibitor that can hinder DHA repair. DNA damage repair is an important mechanism of drug resistance and one of the main causes of metastasis and recurrence after chemotherapy. Etoposide, as a chemotherapy drug included in the "Chinese Medical Association Guidelines for Clinical Diagnosis and Treatment of Lung Cancer", has been widely used in clinical practice. Like other chemotherapy drugs, etoposide also has serious toxic side effects, including bone marrow suppression, mucositis, cardiotoxicity, nephrotoxicity, hair loss and immunosuppression.

[0004] Dihydroartemisinin (DHA) is an active metabolite of artemisinin and its derivatives (ARTs). It is an effective clinical drug widely used to treat malaria. In addition, DHA has been found to show potential anti-tumor effects in cancer treatment. Studies have shown that DHA exerts anti-cancer effects through multiple molecular mechanisms such as inducing cell apoptosis, inhibiting proliferation, inhibiting tumor metastasis, inhibiting angiogenesis, promoting immune function, inducing autophagy and endoplasmic reticulum (ER) stress. At the same time, studies have shown that DHA reduces the side effects caused by cisplatin treatment and reverses cisplatin resistance. It is worth noting that DHA has a synergistic anti-tumor effect with a variety of clinical drugs.

[0005] Chrysin (CRS) is a natural flavonoid compound found in honey and a variety of plant extracts. It has a wide range of pharmacological and physiological activities. Given its wide distribution and relatively low toxicity, chrysin is considered a promising drug for the treatment of diseases. In recent years, chrysin has attracted much attention for its anti-tumor and antioxidant activities and its protective effects on allergic inflammation. Chrysin administration can improve the status of lipid peroxidation and antioxidants, thereby regulating the oxidant / antioxidant balance in cancer. The main anti-cancer mechanisms of chrysin include inhibiting cell proliferation, inhibiting inflammation, and inducing cell apoptosis. In addition, studies have found that chrysin can be used in combination with the clinical chemotherapy drug pyrotinib to exert an anti-breast cancer effect.

[0006] Topoisomerase II (TOP2) is a nuclear protein required for DNA replication and cell division. Therefore, TOP2 has become the main cellular target of many of the most widely used and effective chemotherapy drugs, including doxorubicin, etoposide and mitoxantrone. There are two different genetic isoforms of topoisomerase II (TOP2A and TOP2B), which are expressed and regulated differently in living cells. TOP2B is expressed in almost all quiescent cells of all tissues throughout the cell cycle. TOP2A is usually expressed at high levels in rapidly proliferating and growing cells. DNA topoisomerase II (TOP2A) is closely related to the occurrence, invasion, treatment and prognosis of malignant tumors. It regulates the biological behavior of tumors by participating in various biological processes such as cell cycle and apoptosis. It is also closely related to the targets of tumor chemotherapy drugs.

[0007] Traditional Chinese medicine has the advantages of large production, low toxicity and side effects, and multiple targets. In the treatment of lung cancer, traditional Chinese medicine has been shown to prolong survival rates with fewer toxic side effects. Traditional Chinese medicine combined with chemotherapy drugs is an adjuvant treatment that can be used with traditional Chinese medicine. A large number of studies have shown that traditional Chinese medicine can not only relieve the clinical symptoms of cancer patients and improve their quality of life, but also improve the effects of chemotherapy and radiotherapy, and reduce the adverse reactions and complications caused by radiotherapy or targeted therapy. With the deepening of cancer treatment, patients' drug resistance is gradually increasing. Traditional Chinese medicine combined with chemotherapy plays a synergistic role in improving the effect of chemotherapy and reducing its toxic side effects. Summary of the invention

[0008] In order to overcome the deficiencies of the prior art, the present invention provides the use of dihydroartemisinin combined with chrysin in the preparation of a product for improving the sensitivity of tumor cells to TOP2A inhibitors.

[0009] The above-mentioned object of the present invention is achieved through the following technical scheme: application of dihydroartemisinin combined with chrysin in the preparation of a product for improving the sensitivity of tumor cells to TOP2A inhibitors.

[0010] Furthermore, the dihydroartemisinin is combined with chrysin in the preparation of a product for increasing the sensitivity of tumor cells to TOP2A inhibitors, and the TOP2A inhibitors are specifically used to hinder DNA repair.

[0011] Furthermore, the TOP2A inhibitor is etoposide.

[0012] Furthermore, the concentration ratio of dihydroartemisinin to chrysin is dihydroartemisinin: chrysin = 20 μM: 25 μM or 11 μM: 30 μM.

[0013] Furthermore, the tumor is lung cancer.

[0014] In a further preferred embodiment of the present invention, the lung cancer is small cell lung cancer and non-small cell lung cancer.

[0015] Another object of the present invention is to protect a pharmaceutical composition for increasing the sensitivity of tumor cells to TOP2A inhibitors, comprising a TOP2A inhibitor, dihydroartemisinin and chrysin.

[0016] Furthermore, the TOP2A inhibitor in the pharmaceutical composition is etoposide, and the ratio of dihydroartemisinin: chrysin: etoposide is 20 μM: 25 μM: 5.22 μM or 11 μM: 30 μM: 5.221 μM.

[0017] Furthermore, the pharmaceutical composition also includes excipients acceptable in the pharmaceutical field.

[0018] The beneficial effects of the present invention compared with the prior art are: the present invention has found that the combined use of dihydroartemisinin combined with chrysin and TOP2A inhibitors can increase the sensitivity of TOP2A inhibitors, reduce the dosage, and prolong the treatment time. The pharmaceutical composition of the present invention is based on the inhibition of TOP2A expression by dihydroartemisinin combined with chrysin, causing DNA damage and promoting the occurrence of apoptosis of lung cancer cells, thereby increasing the sensitivity of lung cancer cells to TOP2A inhibitors, enhancing the therapeutic effect of the drug, and can be used to treat the occurrence of TOP2A inhibitor resistance. It can be used to treat the occurrence of TOP2A inhibitor resistance. The present invention proposes a new solution for the existing TOP2A inhibitor resistance, fully utilizing and extending the life cycle of existing drugs, which is more in line with the current level of the pharmaceutical industry and the current status of medical insurance investment in terms of pharmaceutical economics principles. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0020] Figure 1Schematic diagram of the effect of artemisinin combined with chrysin in enhancing the inhibitory activity of etoposide on lung cancer cells in different lung cancer cell lines (* indicates that different concentration groups are compared with the DHA half-to-lethal concentration treatment group; # indicates that different concentration groups are compared with the CRS half-to-lethal concentration treatment group), the final concentration of dihydroartemisinin is 20μM / 11μM; the final concentration of chrysin is 25μM / 30μM; the final concentration of etoposide is 5.22μM (# / *P<0.05, ## / **P<0.01, ### / ***P<0.001, #### / ****P<0.0001, n=3);

[0021] Figure 2 Schematic diagram of the effects of combined drugs and etoposide, dihydroartemisinin and chrysin on the activity of normal human lung epithelial cells (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3);

[0022] Figure 3 Schematic diagram of the effect of the combined drug on TOP2A expression and DNA damage (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3);

[0023] Figure 4 Schematic diagram of the effect of the combined drug on cell apoptosis (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3). DETAILED DESCRIPTION

[0024] The present invention is described in detail below by specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0025] Materials and instruments

[0026]

[0027]

[0028] Example 1

[0029] A pharmaceutical composition for improving the sensitivity of tumor cells to TOP2A inhibitors, the drug comprising dihydroartemisinin, chrysin and a TOP2A inhibitor, the final concentrations of the TOP2A inhibitor, dihydroartemisinin and chrysin in the pharmaceutical composition and the types of antibiotics are shown in Table 1.

[0030] Table 1

[0031]

[0032]

[0033] The preparation method of the pharmaceutical composition of this embodiment is:

[0034] (1) Using a 1 / 10,000 scale, weigh 11.77 mg of etoposide and dissolve it in 2 ml of DMSO to prepare a 10 mM stock solution; weigh 142.18 mg of dihydroartemisinin and dissolve it in 5 ml of DMSO to prepare a 100 mM stock solution; weigh 127.12 mg of chrysin and dissolve it in 5 ml of DMSO to prepare a 100 mM stock solution.

[0035] (2) Using 1640 medium containing 10% serum, the etoposide stock solution was diluted to a 20.88 μM working solution; the dihydroartemisinin stock solution was diluted to a 20 μM and 11 μM working solution; and the chrysin stock solution was diluted to a 25 μM and 30 μM working solution.

[0036] (3) Using DMEM low-glucose medium containing 10% serum, the etoposide stock solution was diluted to a 20.88 μM working solution; the dihydroartemisinin stock solution was diluted to a 20 μM and 11 μM working solution; and the chrysin stock solution was diluted to a 25 μM and 30 μM working solution.

[0037] (4) Use 1640 medium containing 10% serum to prepare a working solution of etoposide: dihydroartemisinin: chrysin: 10.44 μM: 20 μM: 25 μM / 5.22 μM: 20 μM: 25 μM / 2.61 μM: 20 μM: 25 μM.

[0038] (5) Use 1640 medium containing 10% serum to prepare a working solution of etoposide: dihydroartemisinin: chrysin: 10.44 μM: 11 μM: 30 μM / 5.22 μM: 11 μM: 30 μM / 2.61 μM: 11 μM: 30 μM.

[0039] (6) Use DMEM low-glucose culture medium containing 10% serum to prepare a working solution of etoposide: dihydroartemisinin: chrysin: 10.44 μM: 20 μM: 25 μM / 5.22 μM: 20 μM: 25 μM / 2.61 μM: 20 μM: 25 μM.

[0040] (7) Use DMEM low-glucose culture medium containing 10% serum to prepare a working solution of etoposide: dihydroartemisinin: chrysin: 10.44 μM: 11 μM: 30 μM / 5.22 μM: 11 μM: 30 μM / 2.61 μM: 11 μM: 30 μM.

[0041] (8) Use DMEM high-glucose culture medium containing 10% serum to prepare 5.22 μM etoposide working solution; dihydroartemisinin: chrysin: 20 μM: 25 μM / 11 μM: 30 μM working solution; etoposide: dihydroartemisinin: chrysin: 5.22 μM: 20 μM: 25 μM / 5.22 μM: 11 μM: 30 μM.

[0042] Example 2

[0043] Effects of etoposide alone, dihydroartemisinin combined with chrysin, and the combination of etoposide, dihydroartemisinin, and chrysin on the activity of different cells

[0044] Implementation method: Lung cancer H446 cells were seeded in a 96-well plate, with a seeding number of about 6.5×10 4 / well, 14 hours after inoculation, treat cells with etoposide 20.88μM; dihydroartemisinin: chrysin: 20μM: 25μM; etoposide: dihydroartemisinin: chrysin: 10.44μM: 20μM: 25μM / 5.22μM: 20μM: 25μM / 2.61μM: 20μM: 25μM, transfer to a 37℃ constant temperature CO2 incubator overnight for 24 hours, then add CCK837℃ constant temperature CO2 incubator for 1 hour, and use a microplate reader to detect absorbance. The absorbance value reflects cell viability. Set up a blank control group without any drug intervention.

[0045] Implementation method: Lung cancer A549 cells were seeded in a 96-well plate, with a seeding number of about 1.0×10 5 / well, 14 hours after inoculation, treat cells with etoposide 20.88μM; dihydroartemisinin: chrysin: 11μM: 30μM; etoposide: dihydroartemisinin: chrysin: 10.44μM: 11μM: 30μM / 5.22μM: 11μM: 30μM / 2.61μM: 11μM: 30μM, transfer to a 37℃ constant temperature CO2 incubator overnight for 24 hours, then add CCK837℃ constant temperature CO2 incubator for 1 hour, and use a microplate reader to detect absorbance. The absorbance value reflects cell viability. Set up a blank control group without any drug intervention.

[0046] Implementation method: Normal human lung epithelial cells BEAS-2B were seeded in a 96-well plate, with a seeding number of about 1.0×10 5 / well, 14 hours after inoculation, treat cells with etoposide 20.88μM; dihydroartemisinin: chrysin: 20μM: 25μM / 11μM: 30μM; etoposide: dihydroartemisinin: chrysin: 5.22μM: 20μM: 25μM / 5.22μM: 11μM: 30μM, transfer to 37℃ constant temperature CO2 incubator overnight treatment for 24 hours, then add CCK837℃ constant temperature CO2 incubator incubation for 1 hour, and use microplate reader to detect absorbance. The absorbance value reflects the cell viability. Set up a blank control group without any drug intervention.

[0047] Table 2 IC values ​​of H446 and A549 after 24h of treatment with dihydroartemisinin combined with chrysin and etoposide alone 50 Concentration

[0048] Drug treatment <![CDATA[IC 50 They are]]> Dihydroartemisinin: Chrysin 20μM:25μM Dihydroartemisinin: Chrysin 11μM:30μM Etoposide 20.88μM

[0049] Table 3 Effect of combined treatment of etoposide, dihydroartemisinin and chrysin on cell viability of H446 after 24h

[0050] Drug treatment Drug concentration Cell viability Etoposide 20.88μM 54.18％ Dihydroartemisinin: Chrysin 20μM:25μM 53.06％ Etoposide + dihydroartemisinin + chrysin 10.44μM:20μM:25μM 38.42％ Etoposide + dihydroartemisinin + chrysin 5.22μM:20μM:25μM 53.46％ Etoposide + dihydroartemisinin + chrysin 2.61μM:20μM:25μM 68.45％

[0051] Table 4 Effect of combined treatment of A549 with etoposide, dihydroartemisinin and chrysin on cell viability after 24h

[0052] Drug treatment Drug concentration Cell viability Etoposide 20.88μM 53.34％ Dihydroartemisinin: Chrysin 11μM:30μM 53.78％ Etoposide + dihydroartemisinin + chrysin 10.44μM:11μM:30μM 42.11％ Etoposide + dihydroartemisinin + chrysin 5.22μM:11μM:30μM 52.78％ Etoposide + dihydroartemisinin + chrysin 2.61μM:11μM:30μM 68.16％

[0053] Table 5 Effects of different drug combination concentrations on cell viability of normal human lung epithelial cells BEAS-2B after 24 h of treatment

[0054] Drug treatment Drug concentration Cell viability Etoposide 20.88μM Etoposide + dihydroartemisinin + chrysin 5.22μM:11μM:30μM Etoposide + dihydroartemisinin + chrysin 5.22μM:11μM:30μM

[0055] Experimental Results

[0056] CCK8 assay was used to detect cell viability. The experimental results were as follows Figure 1 As shown in Tables 2, 3, 4, and 5, the combination of etoposide, dihydroartemisinin, and chrysin had the same inhibitory effect on the survival rate of lung cancer cells H446 and A549, compared with etoposide, dihydroartemisinin and chrysin, but significantly reduced the effect on the survival rate of normal lung epithelial bronchial cells BEAS-2B; when the IC 50 In these cases, the dosage of the combination drug group was lower than that of the single drug group, indicating that the combination drug is safer.

[0057] Example 3

[0058] The combination of artemisinin and chrysin increases DNA damage in lung cancer cells.

[0059] Implementation method: Lung cancer cells H446 and A549 were inoculated in 24-well plates. After 14 hours of inoculation, the drug concentrations of etoposide: dihydroartemisinin: chrysin: 5.22μM: 20μM: 25μM (H446 cells) / 5.22μM: 11μM: 30μM (A549 cells) were prepared and the cells were treated respectively. The control group was added with 10% serum 1640 medium or 10% serum DMEM medium. After the treatment, the cells were transferred to a cell culture incubator; taken out after 24 hours. 250μL RNAkey Reagent was added to each well, and the lysed cell fluid was collected by blowing and collecting into a 1.5mL Ep tube. 0.1mL volume of chloroform substitute BCP was added to each 1mL RNAkey Reagent, shaken for 15s, allowed to stand at room temperature for 3 minutes, and centrifuged at 12000g 4℃ for 10 minutes. Transfer the upper aqueous phase to a new Eppendorf tube, add 0.5 mL of isopropanol, let stand at room temperature for 10 minutes, centrifuge at 12000 g for 10 minutes at 4°C, discard the supernatant, retain the white precipitate at the bottom, add 1 mL of 75% ethanol to wash the white precipitate, centrifuge at 12000 g for 10 minutes at 4°C, discard the waste liquid, open the lid and turn it upside down on alcohol paper to dry for 10 minutes, add 50 μL of RNA dissolution solution, and bathe at 55°C for 10 minutes. Finally, use an ELISA reader to measure the RNA concentration and quality (quality reference OD 260 / 280 The RNA was reverse transcribed to obtain cDNA, which was then used in qPCR experiments.

[0060] Implementation method: Cell slides were spread on 24-well plates, and then lung cancer cells H446 and A549 were inoculated on the 24-well plates containing cell slides. After 14 hours of inoculation, the cells were treated with drug concentrations of etoposide: dihydroartemisinin: chrysin: 5.22μM: 20μM: 25μM (H446 cells) / 5.22μM: 11μM: 30μM (A549 cells), respectively. The control group was added with 10% serum 1640 culture medium or 10% serum DMEM culture medium. After the treatment, the cells were transferred to a cell culture incubator. After 24 hours, the cells were cultured in a 10% serum 1640 culture medium or a 10% serum DMEM culture medium. The plates were taken out of the box, and 150 μL (the volume of liquid added to the 24-well plate each time is 150 μL) of 4% paraformaldehyde fixative was added to each well at room temperature. After 20 minutes, the plates were washed three times with PBS, each for 5 minutes. 0.3% TritonX-100 was added to permeabilize the cell membrane. After 10 minutes, the plates were washed three times with PBS, each for 5 minutes. Goat serum was added to block the cells. After 1 hour of treatment, TOP2A antibody (antibody dilution concentration was 1:200) was added, and the plates were transferred to a 4°C refrigerator for overnight incubation. The next day, the 24-well plate was removed and washed three times with PBS for 5 minutes each time; DyLight 488 and GoatAnti Mouse IgG (dilution concentration of 1:500) were added, incubated at room temperature for 1 hour, and then washed three times with PBS for 5 minutes each time; 0.6 μg / mL DAPI was added for staining, and after 10 minutes, it was washed three times with PBS for 5 minutes each time; the cell slide in the 24-well plate was removed and inverted on a slide with anti-fluorescence quencher, and finally the slide was transferred to an immunofluorescence microscope for immunofluorescence photography in a dark environment.

[0061] Implementation method: Cell slides were spread on 24-well plates, and then lung cancer cells H446 and A549 were inoculated on the 24-well plates containing cell slides. After 14 hours of inoculation, the cells were treated with drug concentrations of etoposide: dihydroartemisinin: chrysin: 5.22μM: 20μM: 25μM (H446 cells) / 5.22μM: 11μM: 30μM (A549 cells), respectively. The control group was added with 10% serum 1640 culture medium or 10% serum DMEM culture medium. After the treatment, the cells were transferred to a cell culture incubator. After 24 hours, the cells were placed in an incubator. Take out the cells, add 150 μL (the volume of liquid added to the 24-well plate each time is 150 μL) of 4% paraformaldehyde fixative to each well at room temperature, act for 20 minutes, wash three times with PBS, 5 minutes each time; add 0.3% TritonX-100 to permeabilize the cell membrane, act for 10 minutes, wash three times with PBS, 5 minutes each time; add goat serum to block the cells, add γH2A.X antibody (antibody dilution concentration is 1:200) after 1 hour of treatment, and transfer to a 4°C refrigerator for overnight incubation. The next day, the 24-well plate was removed and washed three times with PBS for 5 minutes each time; DyLight 488 and GoatAnti Mouse IgG (dilution concentration of 1:500) were added, incubated at room temperature for 1 hour, and then washed three times with PBS for 5 minutes each time; 0.6 μg / mL DAPI was added for staining, and after 10 minutes, it was washed three times with PBS for 5 minutes each time; the cell slide in the 24-well plate was removed and inverted on a slide with anti-fluorescence quencher, and finally the slide was transferred to an immunofluorescence microscope for immunofluorescence photography in a dark environment.

[0062] Experimental Results

[0063] This experiment used immunofluorescence experiments to detect that the combination of etoposide, dihydroartemisinin, and chrysin can further inhibit the expression of TOP2A at the mRNA level and protein level, and further increase the occurrence of DNA damage. This shows that the combined use of the three drugs has a synergistic effect in inhibiting TOP2A expression and increasing DNA damage.

[0064] Example 4

[0065] The combined action of etoposide, dihydroartemisinin and chrysin induces apoptosis of lung cancer cells.

[0066] Implementation method: The cell slide was spread in a 24-well plate, and then the lung cancer cells H446 and A549 cells were inoculated in the 24-well plate containing the cell slide. After 14 hours of inoculation, the cells were treated with drug concentrations of etoposide: dihydroartemisinin: chrysin: 5.22μM: 20μM: 25μM (H446 cells) / 5.22μM: 11μM: 30μM (A549 cells), respectively. The control group was added with 10% serum 1640 culture medium or 10% serum DMEM culture medium. After the treatment, the cells were transferred to a cell culture incubator; 24 hours later, the cells were taken out from the incubator, the waste liquid was discarded, 5μl AnnexinⅤ-FITC, 5μl PI Staining Solution and 140μl corresponding culture medium were added, and the cells were incubated at room temperature in the dark for 10 minutes. The cell slide in the 24-well plate was taken out and inverted on a slide with anti-fluorescence quencher, and finally the slide was transferred to an immunofluorescence microscope for immunofluorescence photography in a dark environment.

[0067] Implementation method: lung cancer cells H446 and A549 were inoculated in 6-well plates. After 14 hours of inoculation, drug concentrations of etoposide: dihydroartemisinin: chrysin: 5.22μM: 20μM: 25μM (H446 cells) / 5.22μM: 11μM: 30μM (A549 cells) were prepared and the cells were treated respectively. The control group was added with 10% serum 1640 culture medium or 10% serum DMEM culture medium. After the treatment, the cells were transferred to a cell culture incubator; taken out after 24 hours; digested the cells with EDTA-free trypsin, centrifuged at 1800rpm, 4℃ for 5 minutes, and the supernatant was discarded; washed the cells twice with pre-cooled PBS, centrifuged at 1800rpm, 4℃ for 5 minutes each time, and the supernatant was discarded; added 100μl 1×Binding Buffer and gently blown to a single cell suspension; added 5μl AnnexinⅤ-FITC and 5μl PIStaining Solution, blow gently to mix; incubate at room temperature for 10 minutes away from light; add 400μl 1×Binding Buffer, mix gently, and prepare PI single staining and FITC single staining. Normal cells are used to debug the machine, then filtered through a 300-mesh cell screen and transferred to a flow cytometer tube, and finally apoptosis detection is performed in a flow cytometer.

[0068] Experimental Results

[0069] The results of flow cytometry in this experiment showed that the combination of etoposide, dihydroartemisinin and chrysin caused an increase in lung cancer cell apoptosis. This indicates that the combination of the three drugs plays a synergistic role in promoting apoptosis of lung cancer cells.

[0070] The above data results show that the combined use of dihydroartemisinin and chrysin with TOP2A inhibitors can increase the sensitivity of TOP2A inhibitors, reduce the dosage, and prolong the treatment time. The pharmaceutical composition of the present invention is based on the inhibition of TOP2A expression by dihydroartemisinin combined with chrysin, causing DNA damage and promoting the occurrence of lung cancer cell apoptosis, thereby increasing the sensitivity of lung cancer cells to TOP2A inhibitors and enhancing the therapeutic effect of the drug.

[0071] The above-described embodiments are only preferred embodiments of the present invention, but not all feasible embodiments of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. Application of dihydroartemisinin combined with chrysin in the preparation of products that increase the sensitivity of tumor cells to TOP2A inhibitors.

2. The use of dihydroartemisinin combined with chrysin according to claim 1 in the preparation of a product for increasing the sensitivity of tumor cells to TOP2A inhibitors, characterized in that: TOP2A inhibitors are specifically used to hinder DNA repair.

3. The use of dihydroartemisinin combined with chrysin according to claim 1 in the preparation of a product for increasing the sensitivity of tumor cells to TOP2A inhibitors, characterized in that: The TOP2A inhibitor is etoposide.

4. The use of dihydroartemisinin combined with chrysin in the preparation of a product for increasing the sensitivity of tumor cells to TOP2A inhibitors according to claim 1, characterized in that: The concentration ratio of dihydroartemisinin to chrysin is dihydroartemisinin:chrysin=20 μM:25 μM or 11 μM:30 μM.

5. The use of dihydroartemisinin combined with chrysin in the preparation of a product for increasing the sensitivity of tumor cells to TOP2A inhibitors according to claim 1, characterized in that: The tumor is lung cancer.

6. The use of dihydroartemisinin combined with chrysin in the preparation of a product for increasing the sensitivity of tumor cells to TOP2A inhibitors according to claim 5, characterized in that: The lung cancer is small cell lung cancer and non-small cell lung cancer.

7. A pharmaceutical composition for increasing the sensitivity of tumor cells to TOP2A inhibitors, characterized in that: Including TOP2A inhibitors, dihydroartemisinin and chrysin.

8. The pharmaceutical composition for increasing the sensitivity of tumor cells to TOP2A inhibitors according to claim 7, characterized in that: The TOP2A inhibitor in the pharmaceutical composition is etoposide, and the ratio of dihydroartemisinin: chrysin: etoposide is 20 μM: 25 μM: 5.22 μM or 11 μM: 30 μM: 5.221 μM.

9. The pharmaceutical composition for increasing the sensitivity of tumor cells to TOP2A inhibitors according to claim 7, characterized in that: The pharmaceutical composition further comprises excipients acceptable in the pharmaceutical field.

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