Use of an aromatase inhibitor in the preparation of a medicament for treating melanoma

Abstract

The present invention belongs to the field of medicine, and particularly relates to the application of an aromatase inhibitor in the preparation of a medicament for treating melanoma. Experiments have shown that the metabolite Androstane-3β,4β,17β-triol (Triol) produced by formestane in cells can activate ERβ-positive melanoma cells, resulting in cell cycle arrest and inhibited cell growth; moreover, the formestane ointment is convenient to use. Since it is a topical drug, patients have a high acceptance level, can directly reach the tumor site, does not pass through the systemic circulation, and has little side effect after long-term use. It provides a new direction for the treatment of melanoma.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the application of an aromatase inhibitor in the preparation of a medicament for treating melanoma. Background Art

[0002] Cutaneous malignant melanoma is a highly malignant tumor formed by the malignant transformation of melanocytes in the basal layer of the epidermis. Due to its high malignancy, the tumor grows rapidly, is prone to metastasis in the early stage, and has a relatively high fatality rate. Currently, the clinical diagnosis and treatment of CMM mainly rely on extensive surgical resection, supplemented by chemotherapy and / or targeted therapy and / or immunotherapy, as well as necessary radiotherapy. Advanced CMM is less sensitive to the first-line chemotherapy regimen based on alkylating agents (dacarbazine / temozolomide), has poor efficacy, and is prone to drug resistance.

[0003] Aromatase inhibitors are commonly used in the treatment of estrogen-dependent diseases, such as breast cancer. They are divided into steroidal aromatase inhibitors (exemestane, formestane) and non-steroidal aromatase inhibitors (letrozole, anastrozole). Clinical studies have found that steroidal aromatase inhibitors can still work after the failure of non-steroidal aromatase in breast cancer patients, such as formestane. Since formestane still has a good effect after breast cancer patients develop resistance to steroidal drugs, researchers reused the drug by changing its dosage form (the original dosage form was an injection, but it had large side effects), and made formestane into an ointment and directly applied it to the breast, and it reached the tumor site through skin absorption, with remarkable effects.

[0004] However, there is currently no research on the use of aromatase inhibitors in the treatment of melanoma. Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides the application of an aromatase inhibitor in the preparation of a medicament for treating melanoma.

[0006] Preferably, the medicament is a medicament that binds to ERβ.

[0007] Preferably, the medicament is a medicament that promotes the transcriptional activity of ERβ.

[0008] Preferably, the medicament is a medicament that induces the expression of ERβ.

[0009] Preferably, the medicament is a medicament that promotes the nuclear translocation of ERβ.

[0010] Preferably, the medicament is a medicament that inhibits the proliferation of cells with high expression of ERβ.

[0011] Preferably, the medicament is a medicament that blocks the progression of the cell cycle and arrests BLM cells in the G1 / G0 phase.

[0012] Preferably, the drug is a drug that reduces the expression of cell cycle-related proteins CDK4 and CyclinD1 and increases the amount of cyclin-dependent kinase inhibitor p27.

[0013] Preferably, the drug is a drug that activates ERβ.

[0014] Preferably, the aromatase inhibitor is formestane.

[0015] The beneficial effects of the present invention are as follows:

[0016] The present invention has verified through experiments that the metabolite Androstane-3β,4β,17β-triol (Triol) produced by formestane in cells can activate ERβ-positive melanoma cells, resulting in cell cycle arrest and inhibited cell growth; moreover, the formestane ointment is convenient to use. Since it is a topical drug, the patient acceptance is high, it can directly reach the tumor site without passing through the systemic circulation, and the side effects are small after long-term use. It provides a new direction for the treatment of melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is a fluorescence polarization intensity data graph.

[0019] Figure 2 It is an RLU data graph.

[0020] Figure 3 It is a result graph of detecting the expression of ERβ in melanoma cells by Western blot.

[0021] Figure 4 It is a result graph of the expression of ERβ protein in melanoma cells BLM and B16F10.

[0022] Figure 5 It is an immunofluorescence detection result graph.

[0023] Figure 6 It is a cell growth rate data graph.

[0024] Figure 7 It is a cell colony formation graph.

[0025] Figure 8 It is a data graph of the number of cells per well.

[0026] Figure 9 It is a graph of cell number data.

[0027] Figure 10 It is a statistical graph of cell cycle distribution.

[0028] Figure 11 It is a real - shot picture of tumor size.

[0029] Figure 12 It is a graph of mouse body weight and tumor volume data.

[0030] Figure 13 It is a result graph of Western blot analysis of the expression of cell cycle - related proteins.

[0031] Figure 14 It is a graph of the relative expression level of ERβ mRNA.

[0032] Figure 15 It is a graph of relative cell growth rate. Detailed implementation mode

[0033] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other implementation manners obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope protected by the present invention.

[0034] Example 1: Effects of Formestane on ERβ of Melanoma Cells

[0035] (1) The metabolites of formestane (4 - OHA) in cells are 4 - hydroxytestosterone (4 - OHT) and Androstane - 3β,4β,17β - triol (Triol). Based on the competitive hormone - binding experiment of ERβ fluorescence polarization, the ligand - binding domain of rat ERβ labeled with His glutathione s - transferase epitope (His - GST - ERβLbd) has a final concentration of 25 nM. The fluorescently labeled ERβ ligand, fluormoone EL Red, has a final concentration of 2 nM. The competitive test compounds are 4 - OHA, 4 - OHT, Triol and E2. The GraphPad Prism software is used to fit the curve to determine the IC50, and the results are as Figure 1 shown. It can be seen from Figure 1 that the metabolite Triol of formestane can bind to ERβ.

[0036] (2) Further perform the dual-luciferase reporter gene assay. The test compound regulates the transcriptional activity of ERβ at a concentration of 100 nM. Under steroid-free conditions, ERE(3x) regulates the dual-luciferase activity in U2-OS cells. Cells were transiently transfected with pcDNA3.1-ERβ and ERE3-luc2P (firefly luciferase reporter plasmid) and the internal normalization control pRL-TK (Renilla luciferase reporter plasmid). Six hours after transfection, cells were treated with the compounds as shown in the figure. The dual-luciferase activity was detected 48 h after transfection, and the results are as Figure 2 shown. It can be seen from Figure 2 that: The metabolite Triol of Formestane binds to ERβ and promotes its transcriptional activity.

[0037] (3) Extract the proteins of melanoma cell lines BLM, WM115, B16F10, and IGR-39, and detect the expression levels of each cell line by Western blot using the ERβ antibody 14C8. The results are as Figure 3 shown. It can be seen from Figure 3 that: ERβ is expressed in BLM, WM115, and B16F10 cells, while ERβ is hardly expressed in IGR-39 cells.

[0038] (4) After treating BLM and B16F10 cells with 100 nM Formestane and 10 nM DPN for 48 h respectively, collect the samples, extract the proteins, and detect the expression of ERβ. The results are as Figure 4 shown. It can be seen from Figure 4 that: Both Formestane and DPN can induce the expression of ERβ.

[0039] (5) After treating BLM and B16F10 cells with 100 nM Formestane and 10 nM DPN for 48 h respectively. Fix the cells, and detect the expression and localization of ERβ by immunofluorescence. The results are as Figure 5 shown. It can be seen from Figure 5 that: DPN and Formestane promote the nuclear translocation of ERβ.

[0040] The above results indicate that 4-OHA activates the transcriptional activity of ERβ in melanoma cells.

[0041] Example 2: Effects of Formestane on the Growth of Melanoma Cells

[0042] (1) To determine whether Formestane (4-OHA) affects the growth of melanoma cells through ERβ, using the ERβ activator DPN as a positive control, after treating melanoma cells BLM, WM115, B16F10, and IGR-39 with different concentrations of 4-OHA for 72 h, the cell viability was detected by MTT. The results are as Figure 6 shown.

[0043] Then, 1×10 4 BLM and B16F10 cells were seeded onto 6-well plates. After they adhered to the plate, they were treated with drugs for 7 days, and the medium was changed every 2 days. The drug concentrations were as follows: DPN, 10 nM; 4-OHA, 100 nM; ICI, the ERβ antagonist ICI182,780, at a concentration of 1 μM. The results are shown in Figure 7 、 Figure 8 .

[0044] It can be seen from Figures 6 to 8 that 4-OHA significantly inhibited the proliferation of ERβ-highly expressed cells BLM, WM115, and B16F10, had little effect on the proliferation of IGR-39 cells that did not express ERβ, and the ERβ inhibitor ICI182,780 (ICI) could antagonize the effect of 4-OHA.

[0045] Furthermore, after treating BLM cells with 100 nM formestane for 72 h, the cells were fixed, stained with 50 mg / mL propidium iodide, and then treated with 0.2% Triton-X-100 in the dark for 30 min. The cell cycle was analyzed by flow cytometry. The results are shown in Figure 9 、 Figure 10 . It can be seen from Figure 9 、 Figure 10 that 4-OHA and DPN blocked the cell cycle progression, causing BLM cells to arrest in the G1 / G0 phase.

[0046] (2) Five-week-old male BALB / c nude mice or male C57BL / 6 mice were subcutaneously inoculated with BLM and B16F10 cells. When the tumors reached an average volume of 400 mm 3 , the mice (10 mice / group) were respectively smeared with formestane (4-OHA) ointment and excipient cream. It was smeared twice a day, 0.2 mL each time, for 4 consecutive weeks, and the tumor size and mouse body weight were measured once every 3 days. The results are shown in Figures 11 to 12 . The results showed that formestane ointment significantly inhibited the proliferation of melanoma cells and alleviated the weight loss.

[0047] (3) After smearing formestane on mouse melanoma for 4 weeks, the tumor masses were collected, proteins were extracted, and the expression of cell cycle-related proteins was analyzed by Western blot. The results are shown in Figure 13 . It can be seen from Figure 13 that 4-OHA decreased the expression of cell cycle-related proteins CDK4 and CyclinD1 and increased the amount of cyclin-dependent kinase inhibitor p27.

[0048] In summary, in vitro and in vivo experiments both showed that formestane could activate ERβ to inhibit the growth of melanoma cells and shrink tumors.

[0049] Example 3: Formestane activates ERβ to control melanoma growth

[0050] In order to determine whether formestane exerts its anti-melanoma effect by activating ERβ, three shRNAs with different ERβ sequences (named: sh1, sh2, sh3) were used in the melanoma cell line BLM to construct BLM cells with stable knockdown of ERβ. The target sequences are:

[0051] shRNA1: 5'-AGCACGGCTCCATATACAT-3';

[0052] shRNA2: 5'-AGCCACCATGAATATCCAG-3';

[0053] shRNA3: 5'-TGCTGTGATGAATTACAGC-3'.

[0054] First, the efficiency of shRNA knockdown of ERβ was verified, and BLM cells with stable knockdown of ERβ were constructed. The quantitative results are shown in Figure 14 As shown. Figure 14 It can be seen that compared with the control group shCtrl, sh1 and sh2 significantly reduced the expression of ERβ, and sh3 also reduced the expression of ERβ, but not as significantly as sh1 and sh2. Therefore, cell lines with stable expression of sh1 and sh2 were used in subsequent drug treatment experiments.

[0055] The response of ERβ knockdown cells to formestane and DPN was further studied. Figure 15 As shown, stable knockdown of ERβ offset the anti-tumor effects of DPN and formestane, indicating that ERβ expression in melanoma cells is the key to the effect of formestane.

[0056] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Use of an aromatase inhibitor in the preparation of a drug for treating melanoma, characterized in that: The aromatase inhibitor is formestane, and the melanoma is an ERβ-expressing melanoma.

2. The use according to claim 1, characterized in that: The drug induces ERβ expression.

3. The use according to claim 1, characterized in that: The drug promotes ERβ nuclear translocation.

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

5. The use according to claim 1, characterized in that: The drug blocks cell cycle progression and arrests BLM cells in the G1 / G0 phase.

6. The use according to claim 1, characterized in that: The drug reduces the expression of cell cycle-related proteins CDK4 and CyclinD1, and increases the amount of cyclin-dependent kinase inhibitor p27.

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