Application of chelerythrine in preparation of melanoma treatment medicine

Abstract

The invention discloses an application of chelerythrine in preparation of a medicament for treating melanoma. The invention discovers the effect of chelerythrine in treating melanoma, and the chelerythrine can be developed as a means for preventing and treating melanoma by independent or combined medication. Meanwhile, the chelerythrine has a good melanoma metastasis resisting effect, and especially has an inhibiting effect on pulmonary metastasis, so that a new possibility is provided for treating the metastasis of the melanoma.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of chelerythrine in preparing a drug for treating melanoma. Background Art

[0002] Melanoma is a malignant tumor caused by melanocytes. It is a highly malignant tumor on the skin, mucous membranes or pigment membranes that is more common in clinical practice. Malignant melanoma is highly metastatic and can spread from the primary lesion and metastasize to multiple organs, including the lungs, liver, kidneys, brain, etc. Among them, lung metastasis is the most common form of metastasis. Malignant tumors have a serious impact on the physical and mental health and quality of life of patients and their families, but the current treatment options for patients in clinical practice mainly include radiotherapy, chemotherapy, targeted therapy and immunotherapy. However, these treatment methods still have many shortcomings. As traditional treatment methods, radiotherapy and chemotherapy are often accompanied by serious side effects, such as immunosuppression, increased drug resistance and a significant decrease in the quality of life of patients. At the same time, melanoma has a low sensitivity to radiotherapy and chemotherapy, and a high recurrence rate, resulting in poor long-term treatment effects. Although targeted therapy has shown good efficacy in some gene mutation-type melanomas, the problem of drug resistance is still severe because tumor cells are prone to gene mutations, which limits the widespread application of targeted drugs. Although immunotherapy (such as PD-1 inhibitors) has provided breakthrough progress for some patients, its treatment response has obvious individual differences, and its high cost and serious immune-related side effects make it impossible to popularize it to all patients. Therefore, it is urgent to develop a new therapeutic drug with high efficiency and low toxicity, especially for those patients who have become resistant or ineffective to existing therapies.

[0003] In recent years, Chinese herbal medicine extracts have gradually become a hot topic of research due to their multi-target, low toxicity and potential role in inhibiting tumor growth and metastasis. Alkaloid compounds are widely present in Chinese medicinal plants, such as Coptis chinensis, Sophora flavescens, Sanguisorba officinalis, Aconitum carmichaelii, etc. Studies have shown that alkaloid compounds have antioxidant, anti-inflammatory, tumor cell proliferation inhibition and tumor cell apoptosis induction effects, and some natural alkaloids play an important role in cancer prevention and treatment.

[0004] Based on this, the present invention provides the use of chelerythrine in preparing a drug for treating melanoma. Summary of the invention

[0005] One of the purposes of the present invention is to provide the use of chelerythrine in preparing a drug for treating melanoma.

[0006] Furthermore, the drug inhibits the proliferation of melanoma cells.

[0007] Furthermore, the drug inhibits the migration of melanoma cells.

[0008] The second object of the present invention is to provide the use of chelerythrine in the preparation of a drug for inhibiting melanoma metastasis.

[0009] Further, the drug inhibits melanoma lung metastasis.

[0010] The structural formula of chelerythrine described in the present invention is shown below:

[0011]

[0012] The present invention finds the role of chelerythrine in treating melanoma, and can be developed as a means of preventing and treating melanoma alone or in combination. At the same time, chelerythrine has a good anti-melanoma metastasis effect, especially an inhibitory effect on lung metastasis, which provides a new possibility for treating melanoma metastasis.

[0013] The present invention provides a new idea for the treatment of melanoma and the prevention of melanoma metastasis, explores a new use of chelerythrine, and provides a new option for the treatment of malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results show that chelerythrine inhibits the proliferation of B16F10 cells.

[0015] Figure 2 The results show the effect of chelerythrine on the Transwell migration of B16F10 cells.

[0016] Figure 3 The results show the effect of chelerythrine on the scratch repair of B16F10 cells.

[0017] Figure 4 The results are the effects of chelerythrine on lung metastasis of mouse melanoma.

[0018] Figure 5 HE staining of mouse melanoma lung metastasis. DETAILED DESCRIPTION

[0019] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] In the following examples, the experimental results are expressed as mean ± SD, and the data are statistically analyzed using GraphPad 9.5 software. Two-tail Student's t test method is used. **** indicates that there is an extremely significant difference between the two groups, p ≤ 0.0001; *** indicates that there is a very significant difference between the two groups, p ≤ 0.001; ** indicates that there is a significant difference between the two groups, p ≤ 0.01; * indicates that there is a difference between the two groups, p ≤ 0.05.

[0023] Example 1

[0024] 1. CCK-8 cell proliferation assay

[0025] Melanoma cell B16F10 suspension was inoculated in a 96-well plate, 100 μL of cell suspension was added to each well, the cell number range was 2000 / well, and cultured overnight. The next day, after the cells adhered to the wall, the DMEM medium was discarded, and 100 μL / well of medium containing different concentrations of chelerythrine was added again. After 24 hours of culture, 10 μL of CCK-8 reagent was added to each well, and culture was continued for 0.5-4 hours, and the absorbance at 450nm was measured by an enzyme reader.

[0026] like Figure 1 As shown, chelerythrine (CHE) has an inhibitory effect on the proliferation of B16F10 cells.

[0027] 2. Transwell cell migration assay

[0028] Take the melanoma cells B16F10 in good condition and dilute them to a concentration of 1×10 4 / 50μL cell suspension. Add 500μL of 20% serum complete culture medium to the lower chamber in advance, mix 50μL of cell suspension with an equal amount of 0.5μM chelerythrine solution, and add it to the upper chamber. Put it in the incubator and culture it for 24h. Then take out the chamber, wipe off the unmigrated cells in the upper chamber with a cotton swab, fix it with formalin for 15min, stain it with 0.5% crystal violet stain for 30s, and wash the floating color with PBS in time after staining, observe it under a microscope and take pictures. Randomly select five fields of view: upper, lower, left, right, and middle, and get the average number of migrating cells.

[0029] like Figure 2 As shown, chelerythrine (CHE) can delay the migration of B16F10 cells in the Transwell assay, proving that it can inhibit the migration of B16F10 cells.

[0030] 3. Scratch repair experiment

[0031] Melanoma cells B16F10 were inoculated in 6-well plates. The next day, when the cells adhered to the wall and the density reached 70%-80%, a 200μL sterile pipette tip was used to streak the surface layer of the single cells, and then washed once with sterile PBS, replaced with fresh serum-free medium containing different concentrations of chelerythrine, and placed in an incubator for 24 hours. The healing of cell scratches was observed and photographed under a microscope at 0h, 12h, and 24h after scratching. Image J software was used to calculate the cell scratch area and the scratch healing rate.

[0032] like Figure 3 As shown, chelerythrine (CHE) can delay the repair of B16F10 cells in the scratch assay, proving that it can inhibit the migration of B16F10 cells.

[0033] 4. Construction of in vivo melanoma metastasis model by tail vein injection of melanoma cells

[0034] B16F10 cells in the logarithmic growth phase were digested and washed once with PBS. The cells were counted and diluted with pre-cooled PBS to a cell concentration of 2.5 × 10 7 / mL. The cell suspension was placed on ice, and after being suspended and mixed, 200μL of B16F10 cell suspension was inoculated into the tail vein of each mouse. The inoculated experimental mice were randomly divided into two groups: a solvent group; and a 2.5mg / kg administration group of chelerythrine. The drug concentration was configured to be 0.5mg / mL. The drug configuration method was as follows: weigh the drug, first dissolve it fully with 10% DMSO, and then perform subsequent operations. The solvent contained 40% PEG300, 5% Tween 80, and 45% saline. After tail vein inoculation, chelerythrine was given intraperitoneally every two days, and the changes in the physiological state of the mice were monitored and recorded for a total of 23 days.

[0035] like Figure 4 As shown, 23 days after modeling, the tumor burden of melanoma lung metastases in mice was significantly alleviated in the treatment group (n=3) given 2.5 mg / kg chelerythrine (CHE) every two days.

[0036] 5. HE staining

[0037] After melanoma cells were inoculated into the tail vein, the drugs were administered as above. The mice were euthanized 23 days later. One lung tissue of each group of mice was fixed with formalin, embedded in paraffin, and then paraffin sections were made. The paraffin sections were placed in a 65°C oven for 1 hour. After cooling to room temperature, they were dewaxed in the following solutions: xylene, 5min×3; 100% ethanol, 3min×2; 95% ethanol, 3min×2; 70% ethanol, 3min; distilled water, 3min. The paraffin sections were transferred to hematoxylin stain for 1min and rinsed with tap water for 2min; the paraffin sections were transferred to eosin stain for 1min, rinsed with tap water for 10s, and observed under a microscope. The sections were placed in the following solutions for dehydration: 75% ethanol for 3s; 95% ethanol for 3s; 100% ethanol for 3s; xylene for 5min×3; neutral gum was added to the slide for sealing, and observed under a microscope after air drying overnight.

[0038] like Figure 5 As shown, HE staining of lung metastases in mice treated with chelerythrine showed that the tumor area was significantly reduced.

Claims

1. Application of chelerythrine in the preparation of drugs for the treatment of melanoma.

2. The use according to claim 1, characterized in that: The drug inhibits the proliferation of melanoma cells.

3. The use according to claim 1, characterized in that: The drug inhibits the migration of melanoma cells.

4. Application of chelerythrine in the preparation of drugs for inhibiting melanoma metastasis.

5. The use according to claim 4, characterized in that: The drug inhibits melanoma lung metastasis.

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