CN115400134B - MCL-1 inhibitor for treating anaplastic thyroid carcinoma and application thereof - Google Patents

Abstract

The invention relates to the field of thyroid cancer medicines, in particular to an MCL-1 inhibitor for treating anaplastic thyroid cancer, wherein the MCL-1 inhibitor is cinobufaginin-based. The medicine of the invention can be used for treating thyroid cancer, especially undifferentiated thyroid cancer, can significantly promote the death of thyroid undifferentiated cancer cells, and has low side effects.

Description

MCL-1 inhibitor for treating anaplastic thyroid carcinoma and application thereof

technical field

The invention relates to the field of thyroid cancer drugs, in particular to an MCL-1 inhibitor for treating undifferentiated thyroid cancer.

Background technique

Thyroid cancer is on the rise worldwide. In 2015, the incidence of thyroid cancer in China ranked seventh in the incidence spectrum of malignant tumors, accounting for 5.12% of the total. The age-standardized 5-year survival rate of thyroid cancer increased by an average of 5.4 % every three years. From 2007 to 2011, the incidence of thyroid cancer in the United States increased by an average of 4.5% per year, higher than the average for cancer. Common thyroid cancers are divided into four types, papillary thyroid carcinoma (Papillary Thyroid Carcinoma, PTC), thyroid follicular carcinoma (Follicular Thyroid Carcinoma, FTC), medullary thyroid carcinoma (Medullary Thyroid Carcinoma, MTC) and anaplastic thyroid Carcinoma (Anaplastic Thyroid Carcinoma, ATC). ATC is a pathological type with the highest degree of malignancy and extremely poor prognosis among all thyroid cancers, accounting for about 1.3-9.8% of all thyroid cancers. Although its incidence rate is low, its progression is rapid, and it is very easy to involve surrounding structures, causing local infiltration, cervical lymph node metastasis, and even distant metastasis at an early stage. For the treatment of ATC, the currently widely used standard therapy (including surgical resection, radioactive iodine therapy, chemotherapy and its combination therapy) has extremely poor efficacy, and the widely used standard therapy is still doxorubicin combined with radiotherapy and chemotherapy. Drug resistance is easy to develop, and the side effects are great. The average survival period of ATC patients is only 4-6 months. Therefore, it is imminent to explore new drugs and treatment strategies for ATC.

Myeloid cell leukemia-1 (Mcl-1) is an important anti-apoptotic gene in the BCL-2 family. Mcl-1 is overexpressed in some malignant tumors, suggesting that it is related to the occurrence and development of tumors, and can induce tumor cells to resist chemotherapy drugs; down-regulating the expression of MCL-1 protein can promote tumor cell apoptosis, suggesting that Mcl-1 1 is a potential target for cancer therapy. Mcl-1 is also overexpressed in ATC, which may be a marker of poor prognosis in ATC. Studies have shown that Mcl-1 inhibitors, especially S63845, specifically bind to the BH3-binding groove of MCL-1 with high affinity, and efficiently kill MCL-1-dependent cancer cells by activating the BAX/BAK-dependent mitochondrial apoptosis pathway, among which Includes multiple myeloma, leukemia and lymphoma cells. However, there are few reports of Mcl-1 inhibitors in the treatment of ATC, so Mcl-1 small molecule inhibitors are expected to become new anticancer drugs for the treatment of ATC.

Cinobufagin (CB) is a small molecular monomer isolated from the toad skin of the giant bufo bufo. The inventor's research group found that CB can significantly inhibit the activity of ATC cells and induce ATC cell death, which is related to the mitochondrial damage mediated by CB; at the same time, CB can cause a decrease in the expression level of MCL-1 without a significant decrease in MCL-1 mRNA. Therefore, the anti-tumor effect of CB as an MCL-1 inhibitor can provide a new therapeutic entry point and a candidate traditional Chinese medicine monomer for the treatment of ATC.

Contents of the invention

The purpose of the present invention is to provide a novel MCL-1 inhibitor for the treatment of undifferentiated thyroid cancer. Cinobufacino as an MCL-1 inhibitor can significantly promote the death of undifferentiated thyroid cancer cells.

In order to achieve the above-mentioned purpose of the invention, the present invention adopts the following technical scheme: an MCL-1 inhibitor for treating anaplastic thyroid carcinoma, and the MCL-1 inhibitor is cinobufaginin-based.

Preferably, the amount of cinobufaction base is 0.1-2uM. Anaplastic thyroid cancer cell lines include 8505C, KHM-5M, C643, CAL62.

Preferably, the cinobufaction base is extracted from the toad skin of Bufo bufo.

A drug for anaplastic thyroid cancer that includes MCL-1 inhibitors, which are cinobufagin-based.

Application of the cinobufacin base as an MCL-1 inhibitor in the preparation of drugs for the prevention and/or treatment of undifferentiated thyroid cancer.

The MCL-1 inhibitor for treating undifferentiated thyroid cancer adopts the above-mentioned technical solution, and the MCL-1 inhibitor is cinobufacin-based, which can significantly promote the death of undifferentiated thyroid cancer cells with low side effects.

Description of drawings

Figure 1: Schematic diagram of the cytotoxicity of CB in undifferentiated thyroid cancer cell lines (8505C, KHM-5M, CAL62) and the cytotoxicity of MCL-1 inhibitor S63845 in 8505C;

Figure 2: Schematic diagram showing strong binding activity between CB and MCL-1 by molecular docking;

Figure 3: Schematic diagram of the apoptosis rate of 8505C cells by CB and S63845;

Figure 4: Schematic diagram of CB on 8505C cell death;

Figure 5: Schematic diagram of CB on the migration and invasion of 8505C cells;

Figure 6: Schematic diagram of the effect of CB on tumor volume and weight of tumor-bearing nude mice;

Figure 7: Schematic diagram of HE staining of heart, liver, spleen and kidney of animal tissues in different dose groups.

Detailed ways

In the following, the technical solutions in the embodiments of the present invention will be checked and completely described in conjunction with the embodiments of the present invention, and further explanations of the invention will be made. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Given the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The traditional Chinese medicine raw material of the present invention is prepared by extracting from the toad skin of Bufo bufo. The cinobufagin base (Cinobufagin, CB) is a commercially available product, and the manufacturer is Taoshu Biological.

experimental method

1. Obtaining and culturing of undifferentiated thyroid cancer cell lines.

8505C was obtained from the German Culture Collection of Microorganisms (DSMZ), and KHM-5M and CAL62 were purchased from the China Cell Line Resource Bank (Shanghai, China). All cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin in a 37% incubator with 5% CO2.

2. CCK8 cell viability assay was used to detect the cytotoxicity of CB and S63845 in undifferentiated thyroid cancer cell lines.

Anaplastic thyroid cancer cells were seeded into 96-well plates (5000 cells per well) and then cultured in a 37°C, 5% CO2 incubator for 24 hours before treatment. Then, the cells were treated with different concentrations of CB and S63845 for 24 h. At the test point, 10 µl of CCK-8 and 90 µl of RPMI 1640 medium containing 10% FBS were added to each well, and then cultured at 37°C, 5% CO ₂ for nearly 2 h, and then used Synergy LX Multi- Mode Reader (BioTek Instruments, USA) detects OD (optical density) at a wavelength of 450 nm.

3. Molecular docking of CB and MCL-1.

Use PyMOL and AutoDockTool 1.5.6 software to dock key active ingredients and core targets. The structure files (mol2 format) of the key active ingredients and the protein crystal structure (pdb format) of the core target were downloaded from PubChem and RSCB PDB databases respectively.

4. Determination of the mortality rate of anaplastic thyroid cancer cells.

Cell death was detected using Annexin V-FITC and PI kits (Liankebio, China) according to the manufacturer's instructions. ATC cells (1.8×10 ⁵ cells/well) were seeded in 6-well plates, and treated with different drugs after complete attachment. After incubation at 37°C for 24 h, the attached cells and floating cells were collected, washed twice with ice-cold PBS, and then suspended with 1× binding buffer. Cells were labeled with 1µl Annexin V-FITC and 2µl PI, and incubated in the dark at room temperature for 5min. Cell apoptosis was detected by flow cytometry, and cell death rate was measured by Flowjo. The live/dead cell staining kit (Calcein AM·PI) was used to qualitatively detect cell death.

5. Detection of migration and invasion ability of anaplastic thyroid cancer cells.

In order to detect changes in tumor cell migration and invasion abilities, a transwell cell culture chamber (CorningCostar Corp, Cambridge, MA, USA) was used. When detecting changes in invasion ability, coat the upper chamber surface of the bottom membrane of the Transwell chamber with 50mg/L Matrigel™ (BD Biocoat) 1:4 dilution 1 hour in advance, and incubate at 37°C. ATC cells (migration: 2.5 × ¹⁰⁴ cells; invasion: 8 × ¹⁰⁴ cells) were seeded into the upper chamber with serum-free medium, and the lower chamber was filled with 800 ml of 10% FBS medium containing different concentrations of CB drugs. Then, place the 24-well plate in a humidified incubator at 37°C in 5% CO and incubate for 24 h for migration and 48 h for invasion. Use a cotton swab to wipe off cells that have not passed through the filter. Migrated cells on the back of the filter were fixed with 4% PFA (Sigma) for 30 minutes, stained with 0.01% crystal violet for 30 minutes, photographed under an optical microscope and quantified. And quantify with ImageJ software (https://imagej.nih.gov/ij/). All experiments were repeated three times. Colony Formation Assay.

6. In vivo nude mouse xenograft tumor experiment.

Female nude mice (BALB/c, 16-20 g, 3-4 weeks old) were purchased from Shanghai Shrek Biotechnology Company, China, and raised in the Experimental Animal Center of Zhejiang Provincial People's Hospital, China, with a 12-hour day and night cycle and free food and water. Mice were subcutaneously injected with a single 8505C cell suspension (4 × 10 ⁶ cells/100µl). One week after transplantation, the mice were randomly divided into different groups (n = 6/group), treated with 5% DMSO + 40% PEG300 + 10% tween80 and 45% ddH2O, 100μl/mouse, CB (2.5,5mg /kg) dissolved in 5% DMSO + 40% PEG300 + 10% tween80 and 45% ddH2O, intraperitoneally injected every day for 14 consecutive days. Tumor volume and body weight were recorded every other day. The formula for calculating xenograft tumor volume (mm3) is: 0.5 × (shortest diameter) 2 × (longest diameter). After the experiment, all mice were sacrificed, and the dissected xenograft tissues were weighed. All animal experiments were performed in accordance with the experimental procedures approved by the Animal Ethics Committee of Zhejiang Provincial People's Hospital (IACUC-A20220051).

7. Western blot experiment.

Cells were collected 24 hours after drug treatment, and were thawed on ice for 10 minutes using PMSF-containing western and IP lysis buffer (No. P0013, China Best Institute of Biotechnology). The BCA (bisdiphenolic acid) protein assay kit (Thermo Fisher Scientific, USA) was used to quantify the total protein concentration. Protein samples were decomposed by SDS-PAGE precast Tris-Gly gel (4-20%, #P0524M, Beyotime Institute of Biotechnology, China), and then transferred to PVDF membrane. Membranes were blocked for 1 h with 1% Tween-20 in TBST containing 5% skim milk. Subsequently, the membrane was incubated with the corresponding primary antibody overnight at 4°C, followed by an appropriate secondary antibody conjugated to HRP for 60 min at room temperature. Membranes were analyzed with the Fdbio-dura ECL kit (#FD8020, FdbioScience, China) and imaged with the ChemiDoc-MP imager (Bio-Rad, USA). Band densities were quantified with ImageJ software.

Experimental results

1. Toxicity of CB and S63845 to cells.

CCK8 was used to detect the proliferation of ATC cells 8505C, KHM-5M, C643, and CAL-62 after treatment with gradient concentrations of CB for 24 hours, and the proliferation of 8505C cells after treatment with S63845 for 24 hours. The results showed that CB and S63845 could induce dose-dependent death of ATC cells , and the same dose of CB is better. In 8505C, CB can kill 50% of the cells when the drug concentration is 50nM, while S63845 only reaches 50% when the drug concentration is about 10000nM.

As shown in Figure 1, it shows that the IC50 of CB in anaplastic thyroid cancer cells are: 8505C: 52.23 nM; KHM-5M: 218.5 nM; CAL-62: 158.0 nM. The above results indicated that the efficacy of CB in 8505C was the best, and that in KHM-5M and CAL-62 was relatively poor. Subsequent use of MCL-1 high-efficiency inhibitor-S63845 in 8505C cells further verified the effect of existing inhibitors. A higher dose of S63845 can induce the same cell death, 4000nM CB can cause more than 90% ATC cell death, while 4000nM S63845 can only cause less than 10% ATC cell death. Therefore, CB is expected to replace S63845 as a new MCL-1 inhibitor.

2. Molecular docking of CB and MCL-1.

As shown in Figure 2A, in the 3D structure, the MCL-1 protein was verified by molecular docking with CB, and it was verified that the binding energy between CB and MCL-1 was ≤ -8.0 kcal/mol, which indicated strong binding activity. This is also true in the 2D structure shown in Figure 2B.

3. Through cell flow cytometry, CB can significantly promote the death of 8505c cells at the same concentration as S63845.

As shown in Figure 3, when the doses of CB and S63845 are the same (0nM, 100nM, 200nM, 400nM), the survival rates of CB in 8505C cells are about 95%, 70%, 50%, and 40% respectively; KHM-5M The survival rates were about 97%, 93%, 90%, and 83% respectively; the survival rates of CAL-62 were about 96%, 94%, 93%, and 92%; and the survival rates of S63845 were 98% when acting on 8505C cells. %, 97%, 97%, around 95%. That is, the same dose of CB can specifically cause more death of 8505C.

4. CB can induce dose-dependent cell death of 8505C cells, and a dose-dependent decrease of Mcl-1.

A 24-well plate was used to inoculate 2×10 ⁵ cells/well of 8505C cells. After 24 hours, different concentrations of CB5 were given for 24 hours, and a control group was set at the same time. Then the treated 8505C cells were stained with Calcein-AM and PI by IF technology, and the results showed that 8505C cells had dose-dependent cell death. And WB can detect the dose-dependent reduction of Mcl-1, PARP cleavage, and increase of CytoC, that is, CB can down-regulate the expression of Mcl-1 protein in ATC cells to promote tumor cell apoptosis. The apoptosis marker PARP was cleaved, and CytoC was also detected to increase, which also proved that CB can induce cell apoptosis.

5. Cell migration and invasion experiments, CB can significantly inhibit the migration and invasion of 8505c cells.

As shown in Figure 5, the Transwell chamber experiment showed that CB could inhibit the migration and invasion of 8505C cells in a dose-dependent manner.

6. In animal experiments, different concentrations of CB groups (2.5mg/Kg, 5mg/Kg) were set to treat anaplastic thyroid cancer in mice. Both low-dose and high-dose CB can significantly inhibit tumor growth, and high-dose CB can, and the higher the dose, the better the effect (Fig. 6A, 6C). Compared with the NC group, the tumor weight and tumor volume of the CB low-dose group and high-dose group were significantly reduced (Fig. 6D, 6E). There was no significant change in the body weight of the mice (Fig. 6B).

7. As shown in Figure 7, there is no difference in HE staining of heart, liver, spleen, and kidney in animal tissues of different dose groups, that is, the results show that the dose of CB has no toxic and side effects on animals.

Claims (4)

1. Use of cinobufagin as MCL-1 inhibitor in the preparation of a medicament for the prevention and/or treatment of undifferentiated thyroid cancer.

2. The use according to claim 1, characterized in that cinobufagin is used in a concentration of 0.1-2uM.

3. The use according to claim 1, wherein the thyroid undifferentiated carcinoma cell line comprises 8505C, KHM-5M, C643, CAL62.

4. The use according to claim 1, wherein cinobufagin is extracted from toad skin of chinese large toad.

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