Application of combined compound in preparation of medicine for treating lung cancer

Abstract

The invention belongs to the technical field of biological medicine application, and discloses application of a combined compound in preparation of a medicine for treating lung cancer, and the combined compound is dihydroartemisinin and chrysin. The invention researches the application of dihydroartemisinin combined with chrysin in treatment of lung cancer for the first time. Experiments prove that compared with a single medication group of dihydroartemisinin and chrysin, the dihydroartemisinin and chrysin combined medication group has the advantages that the survival rate of lung cancer cells can be more obviously inhibited, and the influence on normal cells is smaller; and under the same condition of reaching IC50, the dosage of the combined medication group is lower than that of a single medication group, so that the occurrence of adverse drug reaction is reduced, a good synergistic anti-tumor effect is shown, the combined medication is safe and effective, and a research direction is provided for the development of new drugs and the new use of old drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical application technology, and specifically relates to the application of a combined compound in the preparation of medicines for treating lung cancer. Background Art

[0002] Lung cancer is one of the most common cancers in the world and one of the malignant tumors with the highest morbidity and mortality rates worldwide. According to the histology of cancer cells, lung cancer can be mainly divided into two types: small cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC). NSCLC is the most common subtype, accounting for 85-90% of all lung cancer types. SCLC accounts for about 15% of lung cancer and is the most malignant and the worst prognosis subtype of all lung cancer subtypes. It has two significant characteristics: high metastasis and easy drug resistance. The main means used for lung cancer in the early stage is surgical resection and adjuvant therapy. When it develops to the late stage, the treatment will turn to chemotherapy or radiotherapy. However, almost all traditional chemotherapy drugs have the same limitations, including non-specific targeting, low bioavailability and the development of drug resistance, which limits their efficacy in cancer treatment. Therefore, it is urgent to find new therapeutic drugs to improve the treatment effect of lung cancer patients.

[0003] Dihydroartemisinin (DHA) is an extremely important artemisinin derivative. Compared with artemisinin, it has better water solubility, higher bioavailability, and easier metabolism, which greatly improves its medicinal value. Studies have found that DHA has obvious cytotoxic effects on a variety of tumor cells, but has little toxic side effects on normal tissue cells. DHA can inhibit the invasion and metastasis of cancer cells by regulating the cell cycle, inducing tumor cell apoptosis, inhibiting angiogenesis, and regulating the expression of proteins related to various pathways. It is considered to be an anti-tumor drug with good development prospects. In addition, it is worth noting that DHA has a synergistic anti-tumor effect with a variety of clinical drugs.

[0004] Chrysin (CRS) is a natural flavonoid found in a variety of plant extracts, such as honey and pollen, and has a wide range of biological activities, such as antibacterial, antihypertensive, antiallergic, anti-inflammatory and antioxidant effects. In addition, multiple studies have found that chrysin is also a potent cancer cell inhibitor. For example, chrysin can induce cell death in various cancer types, such as hepatocellular carcinoma, breast cancer, prostate cancer, and pancreatic cancer. In addition, in non-small cell lung cancer (NSCLC) cells, chrysin overcomes chemoresistance to cisplatin and doxorubicin. These findings make chrysin an attractive antitumor agent.

[0005] More and more studies have confirmed the effectiveness of these natural products as anticancer agents in a variety of human cancer experimental models, and a few compounds are undergoing clinical trials. As natural medicines, dihydroartemisinin and chrysin have the advantages of low toxicity and can inhibit a variety of cancers. At the same time, natural medicines are more affordable than synthetic drugs. Not only that, studies have shown that when used in combination with clinical chemotherapy drugs, they can improve the therapeutic efficiency of chemotherapy drugs, providing a new way for the treatment of lung cancer. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides the use of a combined compound in the preparation of a drug for treating lung cancer. The combined use of dihydroartemisinin and chrysin exhibits strong anti-cancer activity at lower drug concentrations, showing good synergistic anti-tumor effects, and thus has good practical application value.

[0007] The above-mentioned object of the present invention is achieved through the following technical scheme: use of a combined compound in the preparation of a drug for treating lung cancer, wherein the combined compound is dihydroartemisinin and chrysin.

[0008] Furthermore, the drug concentration of dihydroartemisinin is 10-160 μM, and the drug concentration of chrysin is 10-160 μM.

[0009] In a further preferred embodiment of the present invention, the drug concentration of dihydroartemisinin is any one of 10 μM, 20 μM, 40 μM, 80 μM and 160 μM.

[0010] In a further preferred embodiment of the present invention, the drug concentration of chrysin is any one of 10 μM, 20 μM, 40 μM, 80 μM and 160 μM.

[0011] Further preferably, the drug concentration ratio of dihydroartemisinin to chrysin is any one of 40μm:50μm, 40μm:25μm, 40μm:3μm, 20μm:50μm, 20μm:25μm, 20μm:13μm, 10μm:50μm, 10μm:25μm, 10μm:13μm, 43μm:30μm, 43μm:15μm, 43μm:8μm, 22μm:30μm, 22μm:15μm, 22μm:8μm, 11μm:30μm, 11μm:15μm, 11μm:8μm.

[0012] In a further preferred embodiment of the present invention, the drug concentration ratio of dihydroartemisinin to chrysin is 20 μM:25 μM.

[0013] In a further preferred embodiment of the present invention, the drug concentration ratio of dihydroartemisinin to chrysin is 11 μM:30 μM.

[0014] Another object of the present invention is to protect a drug containing dihydroartemisinin and chrysin, which comprises dihydroartemisinin, chrysin and one or more pharmaceutically or food-acceptable excipients. The excipients used may be solid or liquid. Solid-state preparations include powders, tablets, dispersed granules, capsules, pills and suppositories. Suitable solid excipients may be magnesium carbonate, magnesium stearate, talcum powder, sugar or lactose. Tablets, powders, pills and capsules are solid dosage forms suitable for oral administration. Liquid-state preparations include solutions, suspensions and emulsions, and embodiments thereof are aqueous solutions or water-propylene glycol solutions for parenteral injection, or oral solutions with added sweeteners and contrast agents. In addition, it can also be made into small water injections for injection, freeze-dried powder injections for injection, large infusions or small infusions.

[0015] The beneficial effects of the present invention compared with the prior art are:

[0016] The present invention studies the application of dihydroartemisinin combined with chrysin in the treatment of lung cancer for the first time. Experiments have shown that the combined use of dihydroartemisinin and chrysin can more significantly inhibit the survival rate of lung cancer cells compared with their single-use groups, while having little effect on normal cells; under the same IC50, the dosage of the combined use group is lower than that of the single-use group, which reduces the occurrence of adverse drug reactions and shows a good synergistic anti-tumor effect, indicating that the combined use is safe and effective, and provides a research direction for the development of new drugs and the new use of old drugs.

[0017] The present invention eliminates the possibility that the combined use of dihydroartemisinin and chrysin will interact and reduce the drug effect of dihydroartemisinin, and finds that the combined use of dihydroartemisinin and chrysin will produce a beneficial effect, which can achieve a better effect of inhibiting cancer cell proliferation while reducing the concentration of the two drugs. The combined use of dihydroartemisinin and chrysin in the present invention can significantly inhibit the proliferation of lung cancer cells.

[0018] The present invention reports for the first time the effect of dihydroartemisinin combined with chrysin on the proliferation of lung cancer cells. The results show that the combined use of dihydroartemisinin can achieve the same effect as the single use of the two drugs while reducing the toxicity of the drugs. At the same time, the present invention also studies the molecular mechanism of cell proliferation related to the use of dihydroartemisinin combined with chrysin. It is found through experiments that the combined use of drugs can upregulate the protein expression of the tumor suppressor gene p53. In addition, flow cytometry experiments have found that after the treatment of the combined drug, the level of cell active oxygen is significantly increased. It should be noted that the anti-cancer mechanism is very complex, and there are still many mechanisms that are not very clear. If the anti-cancer mechanism of dihydroartemisinin combined with chrysin is thoroughly studied, it will help provide new ideas for anti-cancer drug research. 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 1 To investigate the effects of dihydroartemisinin, chrysin alone and their combination on the proliferation of H446, A549 and BEAS-2B cells; two lung cancer cell lines H446, A549 and normal human bronchial epithelial cells BEAS-2B were treated with different dihydroartemisinin, chrysin and their combination, and the survival rates of these three cell lines were detected (* / #P<0.05, ** / ##P<0.01, *** / ###P<0.001, **** / ####P<0.0001, n=3);

[0021] Figure 2 shows the effect of combined use of dihydroartemisinin and chrysin on the expression of p53 protein in H446 and A549 cells; Figure 2A : Immunofluorescence detection of p53 expression level (left: H446, right: A549); Figure 2B : Quantitative statistics of fluorescence intensity (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3);

[0022] Figure 3 To investigate the effect of dihydroartemisinin combined with chrysin on the level of reactive oxygen species in H446 and A549 cells. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1

[0025] Materials and instruments:

[0026]

[0027]

[0028] Effects of dihydroartemisinin and chrysin alone and in combination on the proliferation of lung cancer cells Implementation method: Lung cancer H446 cells were inoculated in 96-well plates, and the inoculation number was 6.5×10 414h after inoculation, cells were treated with 10, 20, 40, 80 and 160μM dihydroartemisinin; 10, 20, 40, 80 and 160μM chrysin; dihydroartemisinin: chrysin: 30μm:50μm, 30μm:25μm, 30μm:13μm, 15μm:50μm, 15μm:25μm, 15μm:13μm, 8μm:50μm, 8μm:25μm, 8μm:13μm, and transferred to a 37℃ constant temperature CO2 incubator for overnight treatment for 24 hours, and then CCK8 was added to the 37℃ constant temperature CO2 incubator for incubation for 1 hour, and the absorbance was detected by an enzyme marker. The absorbance value reflects the cell viability. A blank control group was set without any drug intervention.

[0029] Implementation method: Lung cancer A549 cells were seeded in 96-well plates. The number of cells seeded was 1×10 5 14h after inoculation, cells were treated with 60, 80, 100, 120 and 140μM dihydroartemisinin; 30, 60, 80, 100 and 120μM chrysin; dihydroartemisinin: chrysin: 43μM: 30μM; 43μM: 15μM; 43μM: 8μM; 22μM: 30μM; 22μM: 15μM; 22μM: 8μM; 11μM: 30μM; 11μM: 15μM; 11μM: 8μM, transferred to a 37℃ constant temperature CO2 incubator for overnight treatment for 24 hours, then CCK8 was added to the 37℃ constant temperature CO2 incubator for incubation for 1 hour, and the absorbance was detected by an enzyme marker. The absorbance value reflects the cell viability. A blank control group was set without any drug intervention.

[0030] Implementation method: BEAS-2B lung bronchial epithelial cells were inoculated in 96-well plates, with the number of inoculation exceeding 1×10 5 14h after inoculation, cells were treated with 10, 50, 100, 150 and 200μM dihydroartemisinin; 10, 50, 100, 150 and 200μM chrysin; 80μM and 85μM dihydroartemisinin; 60μM and 100μM chrysin; dihydroartemisinin: chrysin: 20μM: 25μM; 11μM: 30μM, transferred to a 37℃ constant temperature CO2 incubator for overnight treatment for 24 hours, then CCK8 was added to the 37℃ constant temperature CO2 incubator for incubation for 1 hour, and the absorbance was detected by an enzyme marker. The absorbance value reflects the cell viability. A blank control group was set up without any drug intervention.

[0031] Experimental results:

[0032] CCK8 assay was used to detect cell viability. The experimental results were as follows Figure 1As shown in Table 1, the combined use of dihydroartemisinin and chrysin had the same inhibitory effect on the survival rate of lung cancer cells H446 and A549 as compared with their single-drug groups, while the effect on the survival rate of normal lung epithelial bronchial cells BEAS-2B was significantly reduced; at the same 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.

[0033] Among the nine combined drug concentration ratios of dihydroartemisinin and chrysin, the concentration combination that was consistent with the inhibitory effect of single drug use on cell proliferation was 20μm:25μm (H446 cells) / 11μm:30μm (A549 cells). In subsequent experiments, the combined drug group used this preferred concentration.

[0034] Table 1 IC values ​​of H446 and A549 after 24h of treatment with dihydroartemisinin and chrysin alone 50 Concentration

[0035] Drug treatment <![CDATA[IC 50 They are]]> Dihydroartemisinin 80μm Chrysin 100μm Dihydroartemisinin 85μm Chrysin 60μm

[0036] Table 2 Effect of combined treatment of dihydroartemisinin and chrysin on cell viability of H446 after 24h treatment

[0037] Drug treatment Drug concentration Cell viability Dihydroartemisinin 80μm 54.18％ Chrysin 100μm 53.46％ Dihydroartemisinin + Chrysin 40μm:50μm 38.42％ Dihydroartemisinin + Chrysin 40μm:25μm 47.55％ Dihydroartemisinin + Chrysin 40μm:13μm 52.14％ Dihydroartemisinin + Chrysin 20μm:50μm 42.61％ Dihydroartemisinin + Chrysin 20μm:25μm 53.06％ Dihydroartemisinin + Chrysin 20μm:13μm 58.40％ Dihydroartemisinin + Chrysin 10μm:50μm 46.41％ Dihydroartemisinin + Chrysin 10μm:25μm 59.27％ Dihydroartemisinin + Chrysin 10μm:13μm 68.45％

[0038] Table 3 Effect of combined treatment of dihydroartemisinin and chrysin on cell viability of A549 after 24h treatment

[0039] Drug treatment Drug concentration Cell viability Dihydroartemisinin 85μm 52.74％ Chrysin 60μm 53.34％ Dihydroartemisinin + Chrysin 43μm:30μm 42.11％ Dihydroartemisinin + Chrysin 43μm:15μm 48.35％ Dihydroartemisinin + Chrysin 43μm:8μm 55.62％ Dihydroartemisinin + Chrysin 22μm:30μm 49.63％ Dihydroartemisinin + Chrysin 22μm:15μm 60.74％ Dihydroartemisinin + Chrysin 22μm:8μm 62.30％ Dihydroartemisinin + Chrysin 11μm:30μm 53.78％ Dihydroartemisinin + Chrysin 11μm:15μm 64.76％ Dihydroartemisinin + Chrysin 11μm:8μm 68.16％

[0040] Table 4 Effects of dihydroartemisinin, chrysin alone and in combination on cell viability of BEAS-2B cells after 24h treatment

[0041] Drug treatment Drug concentration Cell viability Dihydroartemisinin 80μm 32.05％ Chrysin 100μm 53.02％ Dihydroartemisinin + Chrysin 20μm:25μm 73.72％ Dihydroartemisinin 85μm 27.83％ Chrysin 60μm 63.57％ Dihydroartemisinin + Chrysin 11μm:30μm 77.09％

[0042] Effects of dihydroartemisinin combined with chrysin on the expression of p53 protein in cells

[0043] Implementation method: The cell slides were spread in a 24-well plate, and then lung cancer cells H446 and A549 were inoculated in the 24-well plate containing the cell slides. After 14 hours of inoculation, dihydroartemisinin: chrysin:

[0044] The cells were treated with the drug concentrations of 20 μm: 25 μm (H446 cells) / 11 μm: 30 μm (A549 cells), and the control group was added with 10% serum 1640 medium and 10% serum DMEM medium. After the treatment, the cells were transferred to a cell culture incubator. After 24 hours, the cells were taken out of the incubator, and 150 μL (the volume of liquid added to the 24-well plate each time was 150 μL unless otherwise specified) of 4% paraformaldehyde fixative was added to each well at room temperature. After 20 minutes of action, the cells were washed three times with PBS, each for 5 minutes. 0.3% TritonX-100 was added to permeabilize the cell membrane. After 10 minutes of action, the cells were washed three times with PBS, each for 5 minutes. Goat serum was added to block the cells. After 1 hour of treatment, p53 antibody labeled with 488 fluorescence (antibody dilution concentration was 1:150) was added, and the cells 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. 0.6 μg / mL DAPI was added for staining. After 10 minutes, the plate was washed three times with PBS for 5 minutes each time. The cell slides in the 24-well plate were removed and inverted onto a glass slide with anti-fluorescence quencher. Finally, the glass slides were transferred to an immunofluorescence microscope for immunofluorescence photography in a dark environment.

[0045] Experimental Results

[0046] This experiment used immunofluorescence staining to detect the expression of tumor suppressor gene p53 protein in lung cancer cells after treatment with dihydroartemisinin combined with chrysin. As shown in Figure 2, compared with the control group, the dihydroartemisinin combined with chrysin treatment group significantly promoted the expression of tumor suppressor gene p53, thereby promoting the death of lung cancer cells. This shows that the use of dihydroartemisinin combined with chrysin plays a synergistic role in promoting the death of lung cancer cells.

[0047] Effects of dihydroartemisinin combined with chrysin on intracellular reactive oxygen species levels

[0048] Implementation method: Lung cancer cells H446 and A549 were inoculated in 6-well plates. After 14 hours of inoculation, drug concentrations of dihydroartemisinin: chrysin: 20 μm: 25 μm (H446 cells) / 11 μm: 30 μm (A549 cells) were prepared and the cells were treated respectively. The control group was added with 10% serum 1640 culture medium and 10% serum DMEM culture medium. After the treatment, the cells were transferred to a cell culture incubator. After 24 hours, a final concentration of 10 μM DCFH-DA was prepared (diluted with serum-free culture medium at 1:1000), the 6-well plate was taken out, and the cell culture medium was removed. , add 1 mL of diluted DCFH-DA working solution, incubate in a 37°C cell culture incubator away from light for 30 minutes; take out the 6-well plate, remove the DCFH-DA working solution, wash three times with serum-free medium, add 1 mL of trypsin for 2 minutes, add 1.5 mL of serum-free medium to terminate digestion, transfer the cell suspension into a 1.5 mL centrifuge tube, and then transfer to a centrifuge and centrifuge at 1000 rpm for 3 minutes, discard the waste liquid, take 0.5 mL of serum-free medium to resuspend the cells, filter through a 300-mesh cell screen and transfer to a flow cytometer, and finally perform reactive oxygen detection in a flow cytometer.

[0049] Experimental Results

[0050] In this study, flow cytometry was used to detect the effect of dihydroartemisinin combined with chrysin on the level of reactive oxygen species in lung cancer cells. Figure 3 As shown in the figure, compared with the control group, the group treated with dihydroartemisinin combined with chrysin significantly increased the level of reactive oxygen species in lung cancer cells, further exacerbating the death of lung cancer cells. This shows that the use of dihydroartemisinin combined with chrysin plays a synergistic role in promoting the death of lung cancer cells.

[0051] These data suggest that the combined use of dihydroartemisinin and chrysin is involved in promoting the expression of the tumor suppressor gene p53 and exacerbating the accumulation of reactive oxygen species in lung cancer cells.

[0052] 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. Use of a combined compound in the preparation of a drug for treating lung cancer, characterized in that: The combined compounds are dihydroartemisinin and chrysin respectively.

2. The use of the combined compound according to claim 1 in the preparation of a drug for treating lung cancer, characterized in that: The drug concentration of dihydroartemisinin is 10-160 μM, and the drug concentration of chrysin is 10-160 μM.

3. Use of the combined compound according to claim 2 in the preparation of a drug for treating lung cancer, characterized in that: The drug concentration of the dihydroartemisinin is any one of 10 μM, 20 μM, 40 μM, 80 μM and 160 μM.

4. Use of the combined compound according to claim 2 in the preparation of a drug for treating lung cancer, characterized in that: The drug concentration of chrysin is any one of 10 μM, 20 μM, 40 μM, 80 μM and 160 μM.

5. Use of the combined compound according to claim 2 in the preparation of a drug for treating lung cancer, characterized in that: The drug concentration ratio of dihydroartemisinin to chrysin is any one of 40μm:50μm, 40μm:25μm, 40μm:3μm, 20μm:50μm, 20μm:25μm, 20μm:13μm, 10μm:50μm, 10μm:25μm, 10μm:13μm, 43μm:30μm, 43μm:15μm, 43μm:8μm, 22μm:30μm, 22μm:15μm, 22μm:8μm, 11μm:30μm, 11μm:15μm, and 11μm:8μm.

6. Use of the combined compound according to claim 5 in the preparation of a drug for treating lung cancer, characterized in that: The drug concentration ratio of dihydroartemisinin to chrysin is 20 μM:25 μM.

7. Use of the combined compound according to claim 5 in the preparation of a drug for treating lung cancer, characterized in that: The drug concentration ratio of dihydroartemisinin to chrysin is 11 μm:30 μm.

8. A drug comprising dihydroartemisinin and chrysin, characterized in that: The medicine comprises dihydroartemisinin, chrysin and one or more pharmaceutically or food-acceptable auxiliary materials.

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