Application of coumarin in preparation of anti-lung cancer drugs

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of coumarin in preparation of an anti-lung cancer medicine. It is found for the first time that the coumarin can inhibit proliferation and migration of lung cancer cells, reduce the survival rate of the lung cancer cells and reduce the increase of the volume and the weight of tumor tissues, and finally the anti-tumor effect is effectively achieved, so that a theoretical basis is provided for the development of the medicines for treating the lung cancer by the coumarin, and the application range of the coumarin is expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of gulumbin in the preparation of anti-lung cancer drugs. Background Art

[0002] Lung cancer has become the malignant tumor with the highest morbidity and mortality rate in the world. As the number one cancer killer, the incidence of lung cancer has been on a continuous upward trend in recent years. Due to the lack of early symptoms of lung cancer, most lung cancer patients are already in the advanced stage when diagnosed and lose the opportunity for surgery. Although certain progress has been made in the diagnosis and treatment of lung cancer in recent years, the treatment effect and patient survival rate of lung cancer have improved with the combined use of surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy, but problems such as the heterogeneity and drug resistance of lung cancer still exist, especially for patients with advanced lung cancer, the treatment effect is often not ideal and the prognosis is poor. Therefore, seeking more efficient therapeutic drugs has important clinical significance and social value. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides the use of gulumbin in the preparation of anti-lung cancer drugs.

[0004] The invention provides application of Gulumbin in preparing anti-lung cancer medicine.

[0005] The present invention discovers through experiments for the first time that Gulumbin can reduce the survival rate of lung cancer cells A549, H1299, H292 and H1975, inhibit the clone number and migration ability of A549 and H1299, and proves through mouse experiments that Gulumbin can effectively inhibit the growth of weight and volume of lung cancer tumor tissue, thereby providing a new drug treatment approach for treating lung cancer, and also providing a new application field for Gulumbin.

[0006] In another preferred embodiment, the anti-lung cancer drug is a drug that inhibits the proliferation and migration of lung cancer cells.

[0007] In another preferred embodiment, the anti-lung cancer drug is a drug that inhibits the proliferation and migration of lung cancer cells A549 and H1299.

[0008] In another preferred embodiment, the anti-lung cancer drug is a drug that reduces the survival rate of lung cancer cells.

[0009] In another preferred embodiment, the anti-lung cancer drug is a drug that reduces the survival rate of lung cancer cells A549, H1299, H292 and H1975.

[0010] In another preferred embodiment, the anti-lung cancer drug is a drug that inhibits the growth of volume and weight of lung cancer tumor tissue.

[0011] In another preferred embodiment, the anti-lung cancer drug contains gulumbin as the only active ingredient.

[0012] In another preferred embodiment, the anti-lung cancer drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0013] In another preferred embodiment, the anti-lung cancer drug is an oral preparation or an injection.

[0014] In another preferred embodiment, the oral preparation is any one of tablets, capsules, pills, powders, granules and syrups.

[0015] Compared with the prior art, the present invention has the following beneficial effects.

[0016] The present invention discovers for the first time through experiments that Gurumbin can inhibit the proliferation and migration of lung cancer cells and reduce the survival rate of lung cancer cells, and proves through mouse experiments that Gurumbin can inhibit the increase in tumor tissue volume and weight, thereby providing a new therapeutic drug for lung cancer drugs, and providing a theoretical basis for the development of Gurumbin drugs for the treatment of lung cancer, thereby expanding the application range of Gurumbin. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the cell activity of A549, H1299, H292 and H1975 cells treated with different concentrations of Gulumbin detected by CCK8 method; among them, A is the survival rate of H1299 cells, B is the survival rate of A549 cells, C is the survival rate of H292 cells, and D is the survival rate of H1975 cells.

[0018] Figure 2 The figures are the results of clone colony formation of A549 and H1299 cells treated with different concentrations of Gulumbin; A is the clone colony formation of H1299 cells, B is the clone colony formation of A549 cells, C is the quantitative graph of A, and D is the quantitative graph of B.

[0019] Figure 3 The figures are the migration results of A549 and H1299 cells treated with different concentrations of Gulumbin; A is the migration result of H1299 cells, B is the migration result of A549 cells, C is the quantitative graph of A, and D is the quantitative graph of B.

[0020] Figure 4 The graphs are the weight and volume results of each group of tumor tissues after oral administration of different doses of Gulumbin; among them, A is the graph of each group of tumor tissues, B is the graph of each group of tumor tissues volume results, and C is the graph of each group of tumor tissues weight results. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods; the reagents and materials described are all commercially available unless otherwise specified.

[0023] Currently, lung cancer treatment generally lacks patient specificity and is often prone to produce strong toxic side effects. Drug resistance and adverse reactions largely limit the effectiveness of lung cancer treatment.

[0024] Columbin, also known as Gulunbin, is an orally active diterpene furanolide with anti-inflammatory and anti-trypanosomal effects. Its structure is as shown in formula (1): Formula (1).

[0025] Chinese patent with publication number CN100502859C discloses the use of gulombin, isogulumbin and deoxygulumbine in the preparation of anti-inflammatory drugs, Chinese patent with publication number CN1839825A discloses the use of gulombin, isogulumbin and deoxygulumbine in the preparation of anti-inflammatory drugs, drugs for inhibiting gastric spasm and stomach pain, and Chinese patent with publication number CN1947715A discloses the use of gulombin, isogulumbin and deoxygulumbine in the preparation of drugs for treating arthritis.

[0026] It can be seen that the current application of Gurumbin is mainly for anti-inflammatory purposes, such as arthritis, gastric spasm, stomach pain, etc. The prior art usually mixes it with other agents to prepare anti-inflammatory, gastric spasm and stomach pain drugs. The present invention first discovered through experiments that Gurumbin can inhibit the proliferation and migration of lung cancer cells, thereby achieving an inhibitory effect on lung cancer cells and ultimately effectively playing an anti-tumor role.

[0027] The application of Gulumbin in the preparation of anti-lung cancer drugs is further described below by way of examples.

[0028] Gumbin used in the following examples was purchased from MedChemExpress, catalog number HY-N0389.

[0029] A549, H1299, H292 and H1975 lung cancer cell lines were purchased from American Type Culture Collection (ATCC).

[0030] Example 1 Effect of Gulumbin on the survival rate of lung cancer cells Cell culture: A549, H1299, H292 and H1975 lung cancer cells were cultured in RPMI 1640 medium (Solarbio, #31800) supplemented with 10% (v / v) fetal bovine serum (Gibco, #A5256701).

[0031] The cells were cultured in an incubator at 37°C containing 95% (v / v) air and 5% (v / v) CO2. When the cells grew to about 80%, they were digested with 0.25% (v / v) trypsin containing 0.02% (v / v) EDTA for 2 min, and culture medium containing 10% (v / v) fetal bovine serum was added to terminate the digestion. After centrifugation, the cells were passaged at a volume ratio of 1:3, and the cells in the logarithmic growth phase were cultured for the experiment.

[0032] Experimental reagents and drugs Reagent: CCK8; Drug: Gurumbin, 100 mg of Gurumbin was dissolved in 6.97 mL of dimethyl sulfoxide to prepare a 40 mM Gurumbin stock solution.

[0033] Specific experimental methods: A549, H1299, H292 and H1975 in the logarithmic growth phase were digested with 0.25% (v / v) trypsin containing EDTA, the trypsin was discarded, RPMI 1640 medium containing 10% (v / v) fetal bovine serum was added to stop the digestion and then centrifuged. After centrifugation, the cells were suspended in complete culture medium, counted, and inoculated into 96-well plates with 3000 cells per well, and cultured in an incubator overnight.

[0034] Replace the RPMI 1640 medium containing 0µM, 1µM, 2µM, 5µM, 10µM, 20µM, 30µM, 40µM of Gurumbin mother solution and 10% (v / v) fetal bovine serum, and culture in the incubator for 48 hours. After the incubation, aspirate the medium in the 96-well plate, add the medium mixed with CCK8 and continue incubation for 4 hours, and measure the absorbance at 450nm with an ELISA reader, where the volume ratio of the medium to CCK8 is 10:1. The experiment was repeated three times, and the cell survival rate was calculated by the following formula:

[0035] Cell survival rate = (experimental well - blank well) ÷ (control well - blank well) × 100%.

[0036] The results are as follows Figure 1 As shown in Figure 2, with the increase of gulumbin concentration, the survival rates of A549, H1299, H292 and H1975 lung cancer cells decreased in turn and in a concentration-dependent manner. The median lethality rate IC 50 They were 11.23µM, 7.05µM, 13.88µM and 14.02µM respectively, indicating that Gulumbin could effectively reduce the survival rate of lung cancer cells.

[0037] Example 2 Effect of Gulumbin on the number of lung cancer cell clones Experimental reagents and drugs Reagents: crystal violet, paraformaldehyde; Drug: columbin, 100 mg of columbin was dissolved in 6.97 mL of dimethyl sulfoxide to prepare a 40 mM columbin stock solution.

[0038] Specific experimental methods: A549 and H1299 cells in the logarithmic growth phase were plated in 12-well cell culture plates, with 400 cells in each well. The plates were placed in an incubator and cultured with RPMI 1640 medium containing 0µM, 5µM, 10µM, and 15µM of Gurumbin stock solution and 10% (v / v) fetal bovine serum. Fresh culture medium containing the above-mentioned different concentrations of Gurumbin stock solution was replaced every three days. When clones visible to the naked eye appeared in the culture dish, the culture was terminated. The supernatant was discarded and the cells were carefully washed twice with PBS. The cells were fixed with 4% (v / v) paraformaldehyde for 15 minutes, and then the paraformaldehyde was discarded; the cells were washed twice with PBS, and 1mL of crystal violet was added for staining for 20 minutes, and then the culture dish was washed with clean water, dried, and photographed under a microscope. The experiment was repeated three times.

[0039] The results are as follows Figure 2 As shown, after treatment with gulumbin, the clonal colony-forming ability of A549 and H1299 cells was significantly inhibited, and the number of clones formed was greatly reduced.

[0040] Example 3 Effect of Gulumbin on the Migration Ability of Lung Cancer Cells Experimental reagents and drugs Drug: Gulumbin, take 100 mg of Gulumbin and dissolve it in 6.97 mL of dimethyl sulfoxide to prepare a 40 mM Gulumbin stock solution.

[0041] Specific experimental method: A549 and H1299 cells in the logarithmic growth phase were plated in 6-well cell culture plates, and each well contained 1×10 5 cells and place the culture plate in an incubator. After the cells have filled the culture plate, use a 200µL pipette tip to scratch the cells vertically with a ruler. Wash the cells 1-2 times with PBS to remove the scratched cells. Add RPMI 1640 culture medium containing 0µM, 5µM, 10µM, and 15µM of Gurumbin mother solution and 1% (v / v) fetal bovine serum, and take photos under a microscope to record the initial state of the scratch. Place the culture plate in an incubator at 37°C and 5% CO2, and take photos every 12 hours.

[0042] The results are as follows Figure 3As shown, the migration ability of A549 and H1299 cells was significantly inhibited after treatment with gulumbin.

[0043] Example 4 Effects of Gulumbin on Tumor Tissues in Mice Materials: 18 SPF adult male BALB / c (nu) mice aged 4 to 5 weeks and weighing 20 ± 2 g were purchased from Nanjing Crisbio Animal Co., Ltd. All nude mice were housed in the SPF animal room of the First Affiliated Hospital of Nanchang University, with a temperature of 18°C ​​to 25°C, a humidity of 40% to 60%, and a light-on-dark cycle of 12 hours. Nude mice had free access to food, which was standard laboratory-specific nude mouse feed and water.

[0044] Drug: Gurumbin, 100 mg of Gurumbin was dissolved in 5 mL of dimethyl sulfoxide to prepare a 20 mg / mL Gurumbin stock solution. 1 mL of the stock solution was added to 9 mL of corn oil and mixed evenly to obtain a 2 mg / mL clear Gurumbin solution.

[0045] Specific experimental methods: The experiment was conducted after the nude mice were adaptively fed for 1 week. Mice in good condition and with similar weight were randomly divided into 3 groups, each with 6 mice, including control group, treatment group 1 and treatment group 2. A549 cells with good logarithmic growth phase were used for tumor transplantation experiments. After cell digestion and centrifugation, the cells were counted under a microscope. The counted cells were resuspended in sterile PBS and fixed to 1×10 7 / mL. Use a sterile 1mL syringe to draw 0.1mL of the A549 cell resuspension and inoculate it subcutaneously in the right axilla of BALB / c nude mice. During the experiment, nude mice were gavaged every day for 21 days. The control group was gavaged with normal saline, and treatment groups 1 and 2 were gavaged with the above-mentioned Gulumbin solution. The dosage of treatment group 1 was 5mg / kg of Gulumbin, and the dosage of treatment group 2 was 10mg / kg of Gulumbin. After 21 days, the nude mice were euthanized and the tumor tissue was removed. The weight of the tumor tissue was weighed, and the size of the tumor tissue was measured with a vernier caliper to calculate the volume. The results are as follows Figure 4 As shown, compared with the control group, the weight and volume of tumor tissue in treatment group 1 and treatment group 2 were significantly reduced, and the effect of treatment group 2 was the best.

[0046] From the above experimental results, we can see that Gulumbin can reduce the survival rate of A549, H1299, H292 and H1975 cells, inhibit the number of clones and migration ability of A549 and H1299 cells, and through mouse experiments, we can see that Gulumbin can effectively inhibit the growth of tumor tissue weight and volume, thereby providing a new way for the treatment of lung cancer and also providing Gulumbin with a new application range.

[0047] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Obviously, various changes and modifications may be made to the present invention by those skilled in the art without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such changes and modifications if they fall within the scope of the technical equivalents of the present invention.

Claims

1. Application of Gulumbin in the preparation of anti-lung cancer drugs.

2. The use of Gulumbin in the preparation of anti-lung cancer drugs according to claim 1, characterized in that: The anti-lung cancer drug is a drug that inhibits the proliferation and migration of lung cancer cells.

3. The use of Gulumbin in the preparation of anti-lung cancer drugs according to claim 2, characterized in that: The anti-lung cancer drug is a drug that inhibits the proliferation and migration of lung cancer cells A549 and H1299.

4. The use of Gulumbin in the preparation of anti-lung cancer drugs according to claim 1, characterized in that: The anti-lung cancer drug is a drug that reduces the survival rate of lung cancer cells.

5. The use of Gulumbin in the preparation of anti-lung cancer drugs according to claim 4, characterized in that: The anti-lung cancer drug is a drug that reduces the survival rate of lung cancer cells A549, H1299, H292 and H1975.

6. The use of Gulumbin in the preparation of anti-lung cancer drugs according to claim 1, characterized in that: The anti-lung cancer drug is a drug that inhibits the growth of the volume and weight of lung cancer tumor tissue.

7. The use of Gulumbin in the preparation of anti-lung cancer drugs according to claim 1, characterized in that: The anti-lung cancer drug contains gulumbin as the only active ingredient.

8. The use of Gulumbin in the preparation of anti-lung cancer drugs according to claim 1, characterized in that: The anti-lung cancer drug also includes a pharmaceutically acceptable carrier and / or excipient.

9. The use of Gulumbin in the preparation of anti-lung cancer drugs according to claim 1, characterized in that: The anti-lung cancer drug is an oral preparation or an injection.

10. The use of Gulumbin in the preparation of anti-lung cancer drugs according to claim 9, characterized in that: The oral preparation is any one of tablets, capsules, pills, powders, granules and syrups.

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