Application of melanine in preparation of antitumor drugs

Abstract

The invention discloses an application of melanine in preparation of an antitumor drug. The invention proposes and verifies that the melanine can significantly inhibit the cell proliferation activity of the epidermal growth factor receptor mutant non-small cell lung cancer by inducing ferroptosis for the first time, and significantly inhibit the migration and diffusion of cancer cells and the colony proliferation and tumorigenesis potential of the cancer cells; and a new choice and direction are provided for clinical treatment of the epidermal growth factor receptor mutant non-small cell lung cancer.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and in particular to the application of a purpuric acid alkaloid in the preparation of antitumor drugs. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related deaths worldwide, posing a serious threat to public health. Pathologically, lung cancer is mainly divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC accounting for approximately 80-85% and being the dominant subtype. Common histological types include adenocarcinoma and squamous cell carcinoma. The occurrence of NSCLC is associated with multiple risk factors, including smoking, occupational exposure, a history of chronic lung disease, and genetic susceptibility.

[0003] Among the molecular subtypes of NSCLC, epidermal growth factor receptor (EGFR) mutations are the most common driver gene alterations, especially in Asian populations, where the EGFR mutation rate can reach approximately 40%. EGFR mutations mainly include classic activating mutations such as exon 19 deletion (19del) and exon 21 L858R point mutations, which are key predictive markers of EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy sensitivity. For EGFR mutation-positive NSCLC, EGFR-TKIs have become the first-line standard of care, significantly prolonging patient survival. However, clinical treatment still faces significant challenges: on the one hand, most NSCLC patients are diagnosed at an advanced stage, requiring improved early detection rates; on the other hand, targeted therapy inevitably leads to drug resistance, ultimately causing disease progression, making the exploration of new treatment strategies and drugs crucial.

[0004] Black pine alkaloid is a natural product isolated from the stem of Tripterygium wilfordii, and its biological activity is not yet clear. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide the application of purpuric acid in the preparation of antitumor drugs, especially in the preparation of drugs that induce ferroptosis in non-small cell lung cancer and inhibit its metastasis.

[0006] Technical solution: The application of the black mandin alkaloid described in this invention in the preparation of antitumor drugs.

[0007] Preferably, the CAS number of the black mandin alkaloid is 114542-54-0.

[0008] Preferably, the application is for the preparation of tumor ferroptosis inducing drugs.

[0009] Preferably, the application is for preparing tumor metastasis inhibitory drugs.

[0010] Preferably, the tumor is lung cancer; more preferably, the lung cancer is non-small cell lung cancer; and even more preferably, the non-small cell lung cancer is epidermal growth factor receptor mutant non-small cell lung cancer.

[0011] Preferably, the drug contains melanin or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

[0012] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0013] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention proposes and verifies for the first time that purpuric acid can significantly inhibit the proliferation activity of epidermal growth factor receptor mutant non-small cell lung cancer cells by inducing ferroptosis, and significantly inhibit the migration and spread of cancer cells as well as the proliferation and tumorigenic potential of cancer cell colonies, providing a new option and direction for the clinical treatment of epidermal growth factor receptor mutant non-small cell lung cancer. Attached Figure Description

[0015] Figure 1 Figure 1 shows the results of PC9 cell viability and IC50 values ​​after different treatments. Figure 2 Figure 1 shows the results of HCC827 cell viability and IC50 values ​​after different treatments. Figure 3 Figure 1 shows the results of PC9 cell apoptosis level measurement after different treatments; Figure 4 Figure 1 shows the results of HCC827 cell apoptosis level measurement after different treatments; Figure 5 Figure 1 shows the results of PC9 cell scratch assay after different treatments; Figure 6 Figure 1 shows the results of scratch assays on HCC827 cells after different treatments. Figure 7 Figure 1 shows the results of PC9 cell cycle detection after different treatments; Figure 8 Figure 1 shows the cell cycle detection results of HCC827 cells after different treatments; Figure 9 Figure 1 shows the results of PC9 cell proliferation assay after different treatments. Figure 10 Figure 1 shows the results of HCC827 cell proliferation assay after different treatments. Figure 11 Figure 1 shows the results of colony formation ability assay of PC9 cells after different treatments; Figure 12 Figure 1 shows the results of colony formation ability assay of HCC827 cells after different treatments; Figure 13 Figure 1 shows the results of PC9 cell viability assays after treatment with different combined cell death inhibitors. Figure 14 Figure 1 shows the results of HCC827 cell viability assays after treatment with different combined cell death inhibitors. Figure 15 Figure 1 shows the results of reactive oxygen species (ROS) levels in PC9 cells after different treatments. Figure 16 Figure 1 shows the results of reactive oxygen species (ROS) levels in HCC827 cells after different treatments. Figure 17 Figure showing the results of ferroptosis marker detection in PC9 cells after different treatments; Figure 18 Figure showing the results of ferroptosis marker level detection in HCC827 cells after different treatments; Figure 19 The curves showing the changes in mouse body weight after different treatments; Figure 20 Curves showing changes in tumor volume in mice after different treatments; Figure 21 Images of mouse tumor tissue at different experimental endpoints after different treatments; Figure 22 A graph showing the statistical results of tumor quality in mice after different treatments; Figure 23 Representative images of Ki67 immunohistochemical staining in mouse tumor tissues after different treatments; Figure 24 Figure showing the results of ferrodeogenesis marker level detection in mouse tumor tissues after different treatments. Detailed Implementation

[0016] The technical solution of the present invention will be further described below.

[0017] Example 1: In vitro efficacy verification of regelidine (REG) in non-small cell lung cancer 1. Determination of cell viability and IC50 value after REG treatment PC9 cells (purchased from Wuhan Pronosei Life Sciences Co., Ltd., catalog number CL-0668) or HCC827 cells (purchased from Wuhan Pronosei Life Sciences Co., Ltd., catalog number CL-0094) were administered at a rate of 2×10⁻⁶. 3Inoculate the cells at a density of cells / well in 96-well plates. After culturing for 24 hours, replace the original medium with DMEM complete medium (containing 1‰ DMSO) containing REG (purchased from MedChemExpress LLC., catalog number HY-N6912) at a final concentration of 0, 1.25, 2.5, 5, 10, 20, 50 or 100 µM.

[0018] After 24 h of treatment, each well was replaced with 100 μL of DMEM basal medium containing 10% CCK8 reagent and incubated at 37℃ for 2 h. The absorbance was measured at 450 nm using a microplate reader, and the relative activity was calculated and a cell viability curve was fitted.

[0019] The results are as follows Figure 1 , 2 As shown, the activity of PC9 cells decreased with increasing REG concentration, and the IC50 value of PC9 cells treated with REG for 24 h was 19.97 µM. For HCC827 cells, the CCK8 assay results were consistent with the activity changes of PC9 cells, and the IC50 value of HCC827 cells treated with REG for 24 h was 17.43 µM.

[0020] 2. Determination of apoptosis levels after REG treatment PC9 cells or HCC827 cells were used at a rate of 2 × 10⁶ cells per well. 4 Inoculate at a density of 100 cells / well in 6-well plates, incubate overnight until adherence, then replace the original medium with DMEM complete medium containing 10 or 20 µM REG (both containing 1‰ DMSO) or DMEM complete medium containing 1‰ DMSO, and perform routine culture treatment.

[0021] Cells were collected 24 h after treatment and stained using the Annexin V-PE / 7AAD apoptosis detection kit (catalog number A213-01). The stained cells were analyzed by flow cytometry to quantify the level of apoptosis.

[0022] The results are as follows Figure 3 , 4As shown, compared with the DMSO control group, the apoptosis level of PC9 cells in the REG 10 μM treatment group (REG 10) was significantly increased, and the apoptosis level in the REG 20 μM treatment group (REG 20) was further increased, showing a concentration-dependent effect. This indicates that REG can effectively induce apoptosis in PC9 cells, and the apoptosis-inducing effect increases with increasing drug concentration. Similarly, in HCC827 cells, the apoptosis rate of HCC827 cells in the DMSO control group was at a low level, while the apoptosis level in the REG treatment group was significantly higher than that in the control group. This preliminarily confirms that REG also has a significant apoptosis-inducing effect on HCC827 cells, and this effect increases with increasing concentration, which is consistent with the experimental results of PC9 cells. This indicates that REG can exert a therapeutic effect on EGFR-mutant non-small cell lung cancer cells by inducing apoptosis.

[0023] 3. Cell scratch assay after REG treatment PC9 cells or HCC827 cells were used at a rate of 2 × 10⁶ cells per well. 4 The cells were seeded at a density of 1 cell per well in 6-well plates. Once the cell density reached 70%, cells were scratched in each well using a 10 µL pipette tip. The cells were washed with PBS to remove any detached cells. The original medium was then replaced with DMEM complete medium containing 10 or 20 µM REG (both containing 1‰ DMSO) or DMEM complete medium containing 1‰ DMSO, and the cells were cultured as usual.

[0024] After 24 h of treatment, the cells were cultured in DMEM medium containing 1% FBS for another 48 h. After the culture was completed, the cells in each group were observed and images were collected under an optical microscope at 72 h after scratching.

[0025] The results are as follows Figure 5 , 6 As shown, compared with the DMSO control group, the scratch healing rate of PC9 cells treated with REG 10 μM was significantly reduced, and cell migration was significantly inhibited. The scratch healing rate of the REG 20 μM treatment group was further reduced, and the inhibitory effect on cell migration was more significant, indicating that REG can inhibit the scratch healing ability of PC9 cells in a concentration-dependent manner, that is, significantly inhibit the migration ability of this cell. Similarly, in HCC827 cells, REG has a significant inhibitory effect on cell migration. Compared with the control group, the scratch healing rate of cells treated with REG 10 μM was significantly slower. The scratch healing rate of the REG 20 μM treatment group was further reduced than that of the 10 μM group, and cell migration was more strongly inhibited, indicating that REG can also effectively block the migration process of HCC827 cells.

[0026] 4. Cell cycle detection after REG treatment PC9 cells or HCC827 cells were used at a rate of 2 × 10⁶ cells per well. 4 Inoculate at a density of 100 cells / well in 6-well plates, incubate overnight until adherence, then replace the original medium with DMEM complete medium containing 10 or 20 µM REG (both containing 1‰ DMSO) or DMEM complete medium containing 1‰ DMSO, and perform routine culture treatment.

[0027] Cells were collected 24 h after treatment and stained using the Novizan Cell Cycle Detection Kit (catalog number AC101-01). The stained cells were analyzed by flow cytometry, and the cell cycle was quantitatively analyzed using FlowJo software.

[0028] The results are as follows Figure 7 , 8 As shown, compared to the DMSO control group, the cell cycle distribution of PC9 cells (G0 / G1 phase ratio of 57.5%) increased to 63.8% and 73.0% after treatment with 10 μM and 20 μM REG, respectively. This preliminarily indicates that PC9 cells can be arrested in the G0 / G1 phase, and the arrest effect increases with increasing concentration, suggesting that the drug can inhibit PC9 cell proliferation by interfering with cell cycle progression. Similarly, in HCC827 cells, compared to the DMSO control group, the REG-treated group showed G0 / G1 phase arrest, with the G0 / G1 phase ratio increasing from 50.9% in the control group to 54.9% (REG 10) and 57.5% (REG 20). The cell cycle arrest effect of the 20 μM REG treatment group was more significant than that of the 10 μM group, confirming that REG can induce cell cycle arrest in HCC827 cells in a concentration-dependent manner, interfering with the normal cell proliferation cycle and thus inhibiting cell division and proliferation.

[0029] 5. Assay for cell proliferation capacity after REG treatment PC9 cells or HCC827 cells were used at a rate of 2 × 10⁶ cells per well. 4 Inoculate at a density of 100 cells / well in 6-well plates, incubate overnight until adherence, then replace the original medium with DMEM complete medium containing 10 or 20 µM REG (both containing 1‰ DMSO) or DMEM complete medium containing 1‰ DMSO, and perform routine culture treatment.

[0030] After 24 h of treatment, 20 μL of EdU reagent (purchased from Novizan, catalog number A413-01) was added to each well, and the mixture was incubated at 37 °C for 2 h. After fixation with 4% paraformaldehyde solution at room temperature for 15 min, the mixture was permeated with Beyotime immunostaining permeabilization solution (catalog number P0096) at room temperature for 20 min. Then, Click reaction solution (purchased from Novizan, catalog number A413-01) was added, and the mixture was incubated in the dark for 30 min. The slides were then mounted with Beyotime anti-fluorescence quenching mounting solution (catalog number P0131), and the slides were observed and images were acquired using a fluorescence microscope.

[0031] The results are as follows Figure 9 , 10 As shown, the DMSO control group PC9 cells had a high EdU positivity rate and vigorous proliferation activity. After treatment with 10 μM and 20 μM gentamicin, the proportion of positive cells decreased significantly in a concentration-dependent manner, demonstrating the potent inhibitory effect of REG on the proliferation of PC9 cells. In HCC827 cells, REG also significantly reduced proliferation activity, and the inhibitory effect increased with increasing concentration, consistent with the PC9 cell experiments. These experiments further confirmed that REG has a significant inhibitory effect on the proliferation of EGFR-mutant non-small cell lung cancer cells.

[0032] 6. Assay for cell colony formation ability after REG treatment PC9 or HCC827 cells were seeded at a density of 500 cells / well in 6-well plates and cultured overnight. After adhesion, the original medium was replaced with DMEM complete medium containing 10 or 20 µM REG (both containing 1‰ DMSO) or DMEM complete medium containing 1‰ DMSO, and then cultured as usual.

[0033] After 24 hours of treatment, the medium was replaced with fresh DMEM complete medium without REG, and the medium was replaced every 2 days. After 14 days of culture, the medium was stained with Beyotime crystal violet reagent (product number C0121-100ml), and the images were observed and acquired under a microscope.

[0034] The results are as follows Figure 11 , 12 As shown, compared with the control group, the number of cell colonies formed in the REG 10 μM treatment group was significantly reduced, and the REG 20 μM treatment group showed a more significant inhibitory effect on cell colony formation, indicating that REG can inhibit the colony formation ability of PC9 cells in a concentration-dependent manner, blocking cell colony proliferation and tumorigenic potential. In addition, REG also has a strong inhibitory effect on the colony formation ability of HCC827 cells. Compared with the control group, REG can inhibit the cell colony formation ability in a dose-dependent manner, confirming that the drug can also effectively reduce the in vitro tumorigenic ability of HCC827 cells.

[0035] 7. Cell viability assay after combined treatment with REG and cell death inhibitors PC9 cells or HCC827 cells at 2×10 3 Seeds were planted at a density of 10 μg / well in 96-well plates. After 24 h of incubation, the medium was divided into two categories: DMEM complete medium containing 10 µM REG, or DMEM complete medium containing 10 µM REG and 5 µM Fer-1 (purchased from MedChemExpress LLC., catalog number HY-100579), or DMEM complete medium containing 10 µM REG and 5 µM zVAD (purchased from MedChemExpress LLC., catalog number HY-16658B), or DMEM complete medium containing 10 µM REG and 5 µM NSA (purchased from MedChemExpress LLC., catalog number HY-100573), or DMEM complete medium containing 10 µM REG and 5 mM 3-ME (purchased from MedChemExpress LLC., catalog number HY-19312), or DMEM complete medium containing 10 µM REG and 5 mM 3-ME (purchased from MedChemExpress LLC., catalog number HY-19312). Use µM REG and 5 µM BAY (purchased from MedChemExpress LLC., catalog number HY-13453) in DMEM complete medium (both containing 1‰ DMSO), or replace the original medium with DMEM complete medium containing 1‰ DMSO, and perform routine culture treatment.

[0036] After 24 h of treatment, each well was replaced with 100 μL of DMEM basal medium containing 10% CCK8 reagent and incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader, and the relative activity was calculated.

[0037] The results are as follows Figure 13 , 14 As shown, compared with the cell viability level of the REG treatment group, the activity of PC9 cells significantly increased when combined with ferroptosis inhibitors, and there was no significant difference in activity compared with the DMSO control group. The cell viability of other inhibitor combination treatment groups all recovered compared with the REG treatment group alone, but the recovery level of the ferroptosis inhibitor treatment group was the strongest, indicating that REG mainly exerts its killing effect on PC9 cells by inducing ferroptosis. Similar experimental results were also detected in HCC827 cells. The above experiments preliminarily indicate that REG inhibits the activity of EGFR-mutant non-small cell lung cancer mainly by inducing ferroptosis.

[0038] 8. Determination of reactive oxygen species (ROS) levels after REG treatment PC9 cells or HCC827 cells at 2×10 4Inoculate at a density of 100 cells / well in 6-well plates, incubate overnight until adherence, then replace the original medium with DMEM complete medium containing 10 or 20 µM REG (both containing 1‰ DMSO) or DMEM complete medium containing 1‰ DMSO, and perform routine culture treatment.

[0039] After 24 h of treatment, the cells were washed with pre-cooled PBS, then incubated with a 5 μM DCFH-DA probe (purchased from MedChemExpress LLC., catalog number HY-D0940) at 37 °C for 30 min, and detected and quantified by flow cytometry.

[0040] The results are as follows Figure 15 , 16 As shown, REG can significantly increase ROS levels in PC9 cells in a dose-dependent manner; similar experimental results were obtained in HCC827 cells. These experiments once again confirm that REG can inhibit tumors by inducing ferroptosis.

[0041] 9. Determination of ferroptosis markers after REG treatment PC9 cells or HCC827 cells at 2×10 4 Inoculate at a density of 100 cells / well in 6-well plates, incubate overnight until adherence, then replace the original medium with DMEM complete medium containing 10 or 20 µM REG (both containing 1‰ DMSO) or DMEM complete medium containing 1‰ DMSO, and perform routine culture treatment.

[0042] After 24 h of treatment, cell culture supernatant was collected, and the levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and glutathione (GSH) in the supernatant were detected using the Beyotime Lipid Oxidation (MDA) Detection Kit (Catalog No. S0131S), the Sangon 4-hydroxynonenal (4-HNE) ELISA Kit (Catalog No. D751041), and the Beyotime GSH and GSSG Detection Kit (Catalog No. S0053).

[0043] The results are as follows Figure 17 , 18 As shown, compared with the control group, the levels of MDA and 4-HNE in PC9 cells treated with REG were significantly increased, while the level of GSH was significantly decreased. Moreover, the above changes were concentration-dependent, indicating that REG can effectively induce ferroptosis in PC9 cells. In HCC827 cells, the changes in intracellular ferroptosis markers after REG treatment were similar to those in PC9 cells, with a significant increase in proferoprepancy markers (MDA and 4-HNE) and a significant decrease in antiferoprepancy marker (GSH).

[0044] The above experimental results confirm that ferroptosis is the key mechanism by which REG exerts its anti-tumor effect in EGFR-mutant non-small cell lung cancer.

[0045] Example 2: In vivo efficacy verification of purpuric acid in a mouse model of non-small cell lung cancer. Female Balb / c nude mice, aged 6-8 weeks and weighing 18-20 g, were purchased from the Experimental Animal Center of Nantong University. PC9 cells were administered at a rate of 5 × 10⁻⁶ cells per mouse. 5 One cell was subcutaneously inoculated into the left axilla to create a tumor. The tumor was allowed to grow to approximately 100 mm in size. 3 Tumor-bearing mice were randomly divided into three groups: a saline group, a REG treatment group (20 mg / kg), and a REG + Fer-1 combined treatment group (REG dose 20 mg / kg, Fer-1 dose 10 mg / kg). Drug administration was initiated simultaneously. The first administration was recorded as day 0. Drugs were administered by gavage every two days. The saline group received the same volume of saline. Treatment continued until day 19. Upon reaching the experimental endpoint, the mice were euthanized, and tumor and lung tissues were collected from each group for subsequent analysis of relevant indicators.

[0046] 1. Weight monitoring Starting from the day after drug administration (Day 1), the body weight of mice in each group was measured every 2 days.

[0047] The results are as follows Figure 19 As shown, the changes in body weight of the three groups of mice over time were basically the same, and there was no significant difference between the groups. The treatment concentration of REG selected in the experiment did not produce obvious biological toxicity.

[0048] 2. Evaluation of tumor progression Starting from the day after drug administration (Day 1), the tumor volume of mice in each experimental group was measured every two days, and the length and width of the tumor were recorded. The tumor volume was then calculated using the following formula and statistically analyzed: Tumor volume (mm²) 3 () = length × width × width / 2. At the end of the experiment, weigh the tumor tissue extracted from each mouse.

[0049] Changes in tumor volume, such as Figure 20 As shown, the tumor tissue image at the experimental endpoint is as follows. Figure 21 As shown, the tumor tissue mass at the experimental endpoint was as follows: Figure 22As shown, compared with the saline treatment group, the tumor volume of the nude mice treated with REG was significantly reduced, while the tumor volume of the nude mice in the REG+Fer-1 treatment group recovered and increased. This indicates that REG can achieve the purpose of treating non-small cell lung cancer by inducing ferroptosis. Furthermore, the weight of the tumors in the nude mice in each experimental group was measured after the experiment. The results showed that compared with the saline treatment group, the REG treatment group could significantly inhibit the tumor weight, and this inhibitory effect could be blocked by the ferroptosis inhibitor Fer-1.

[0050] 3. Immunohistochemical detection of Ki67 protein expression in tumor tissue Mouse tumor tissues were fixed in 4% paraformaldehyde solution for 48 h, dehydrated in a gradient manner, embedded in paraffin, and cut into 4 μm sections. Antigen retrieval was performed at 95°C for 20 min using citrate buffer (pH 6.0). Endogenous peroxidase activity was inhibited with 3% hydrogen peroxide solution for 10 min, followed by blocking with 5% bovine serum albumin solution for 1 h. The sections were incubated overnight at 4°C with Ki67 primary antibody (Proteintech, catalog number 27309-1-AP) diluted 1:10000. After washing, the sections were incubated with horseradish peroxidase (HRP) conjugated with secondary antibody (Proteintech, catalog number SA00001-2) diluted 1:500 for 1 h at room temperature. Signal detection was performed using the Beyotime DAB chromogenic reagent kit (catalog number P0202), and cell nuclei were counterstained with hematoxylin. Images were acquired using an optical microscope.

[0051] The results are as follows Figure 23 As shown, after treatment with REG, the expression of Ki67 in tumor tissue decreased significantly, indicating that REG has a good in vivo anti-tumor effect.

[0052] 5. Determination of ferroptosis marker levels in tumor tissue 100 mg of tumor tissue was taken, ground in liquid nitrogen using a mortar and pestle, and then 1 mL of Beyotime Western blotting and IP cell lysis buffer (product number P0013) was added to completely lyse the tissue. After lysing on ice for 30 min, the tissue was centrifuged at 12000 rpm for 30 min to obtain the supernatant. The levels of MDA, 4-HNE, and GSH in the supernatant were detected using the Beyotime Lipid Oxidation (MDA) Detection Kit, the Sangon Biotech 4-Hydroxynonenoic acid (4-HNE) ELISA Kit, and the Beyotime GSH and GSSG Detection Kit.

[0053] The results are as follows Figure 24 As shown, compared with the saline treatment group, REG drug can induce high levels of MDA and 4-HNE and inhibit GSH levels, indicating that REG can significantly induce tumor ferroptosis and ultimately achieve the goal of effectively treating EGFR-mutant non-small cell lung cancer.

Claims

1. The application of a purpuric acid alkaloid in the preparation of antitumor drugs.

2. The application according to claim 1, characterized in that, The CAS number of the black mandin alkaloid is 114542-54-0.

3. The application according to claim 1, characterized in that, The application is for the preparation of tumor ferroptosis inducing drugs.

4. The application according to claim 1, characterized in that, The application is for the preparation of drugs that inhibit tumor metastasis.

5. The application according to any one of claims 1 to 4, characterized in that, The tumor is lung cancer.

6. The application according to claim 5, characterized in that, The lung cancer in question is non-small cell lung cancer.

7. The application according to claim 6, characterized in that, The non-small cell lung cancer mentioned is epidermal growth factor receptor mutant non-small cell lung cancer.

8. The application according to claim 1, characterized in that, The drug contains melanin or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

9. The application according to claim 8, characterized in that, The drug also contains pharmaceutically acceptable excipients.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

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