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

Abstract

The invention relates to the field of medicaments for thyroid cancer, in particular to an MCL-1 inhibitor for treating thyroid undifferentiated carcinoma, wherein the MCL-1 inhibitor is cinobufagin. The medicine of the invention can be used for treating thyroid cancer, especially undifferentiated thyroid cancer, can obviously promote the death of thyroid undifferentiated cancer cells, and has low side effect.

Description

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

Technical Field

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

Background

Thyroid cancer is on an ascending trend worldwide, the incidence of thyroid cancer in 2015 in China is located at 7 th position in the incidence spectrum of malignant tumors, accounting for 5.12% of the whole, and the average 5-year survival rate of thyroid cancer standardized by age is increased by 5.4% every three years. The incidence of U.S. thyroid cancer increased by 4.5% annually on average, higher than the average level of cancer, from 2007 to 2011. Common thyroid cancers are divided into four categories, papillary thyroid carcinoma (Papillary Thyroid Carcinoma, PTC), follicular thyroid carcinoma (Follicular Thyroid Carcinoma, FTC), medullary thyroid carcinoma (Medullary Thyroid Carcinoma, MTC) and undifferentiated thyroid carcinoma (Anaplastic Thyroid Carcinoma, ATC), respectively. ATC is one of the most malignant and very poor prognosis pathological types in all thyroid cancers, and the incidence rate is about 1.3-9.8% of all thyroid cancers. Although the incidence rate is low, the progress is rapid, local infiltration and cervical lymph node metastasis caused by surrounding structures are extremely involved, and distant metastasis can occur even in early stage. For the treatment of ATC, the standard treatment (including surgical excision, radioiodination therapy, chemotherapy and combined treatment thereof) which is widely applied at present has extremely poor curative effect, the standard treatment which is widely applied is still combined with the radiotherapy and the chemotherapy of doxorubicin, the treatment is extremely easy to generate drug resistance, the side effect is extremely large, and the average survival time of ATC patients is only 4-6 months. Therefore, it is urgent to explore new drugs and therapeutic strategies for ATC.

Myeloid leukemia-1 (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 involved in the development and progression of tumors and can induce tumor cells to exert a resistant effect on chemotherapeutic drugs; and down-regulating MCL-1 protein expression can promote tumor cell apoptosis, which suggests that MCL-1 is a potential target for tumor treatment. Mcl-1 is also overexpressed in ATC, possibly a marker of poor ATC prognosis. Studies have shown that Mcl-1 inhibitors, particularly S63845, bind specifically to the BH3 binding groove of Mcl-1 with high affinity, killing Mcl-1 dependent cancer cells, including multiple myeloma, leukemia and lymphoma cells, with high efficacy by activating the BAX/BAK dependent mitochondrial apoptosis pathway. However, mcl-1 inhibitors have been rarely reported in ATC treatment, and thus Mcl-1 small molecule inhibitors are expected to be new anticancer agents for ATC treatment.

Cinobufagin (CB) is a small molecular monomer isolated from the cutis Bufonis of chinese large toads. Earlier studies by the present inventors have found that CB is capable of significantly inhibiting ATC cell activity, inducing ATC cell death, and is associated with CB-mediated mitochondrial damage; while CB causes a decrease in the level of MCL-1 expression without a significant decrease in MCL-1 mRNA. Therefore, CB as an anti-tumor effect of the MCL-1 inhibitor can provide a new treatment access point and candidate traditional Chinese medicine monomers for the treatment of ATC.

Disclosure of Invention

The invention aims to provide a novel MCL-1 inhibitor for treating thyroid undifferentiated carcinoma, and cinobufagin can be used as the MCL-1 inhibitor to obviously promote the death of thyroid undifferentiated carcinoma cells.

In order to achieve the above object, the present invention adopts the following technical scheme: an MCL-1 inhibitor for use in the treatment of thyroid undifferentiated carcinoma, the MCL-1 inhibitor being cinobufagin.

Preferably, cinobufagin is used in a concentration of 0.1 to 2uM. Thyroid undifferentiated carcinoma cell lines include 8505C, KHM-5M, C643, CAL62.

Preferably, cinobufagin is extracted from cutis Bufonis of Bufo siccus sinensis.

A medicament for use in thyroid undifferentiated carcinoma comprising an MCL-1 inhibitor, the MCL-1 inhibitor being cinobufagin.

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

The MCL-1 inhibitor for treating the thyroid undifferentiated carcinoma adopting the technical scheme has the advantages that the MCL-1 inhibitor is cinobufagin, the death of the thyroid undifferentiated carcinoma cells can be obviously promoted, and the side effect is low.

Drawings

Fig. 1: schematic cytotoxicity of CB in thyroid undifferentiated carcinoma cell line (8505C, KHM-5M, CAL 62) and schematic cytotoxicity of MCL-1 inhibitor S63845 in 8505C;

fig. 2: schematic diagram showing that CB and MCL-1 have stronger binding activity by molecular butt joint;

fig. 3: CB. Schematic representation of S63845 versus 8505C apoptosis rate;

fig. 4: schematic of CB versus 8505C cell death;

fig. 5: schematic representation of CB vs 8505C cell migration and invasion;

fig. 6: schematic diagram of the influence of CB on tumor volume and weight of nude mice bearing tumors;

fig. 7: schematic of HE staining of heart, liver, spleen, kidney of animal tissues in different dose groups.

Detailed Description

The technical scheme in the embodiment of the invention is checked and fully described in combination with the embodiment of the invention, and the invention is further explained. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Given the embodiments of the present invention, all other embodiments that would be obvious to one of ordinary skill in the art without making any inventive effort are within the scope of the present invention.

The traditional Chinese medicine is prepared by extracting toad skin of a Chinese large toad, has various effects of tonifying heart, resisting tumor, resisting inflammation, easing pain and the like, has very wide application and development prospect in the aspect of resisting cancer, and is a commercial product, namely Cinobufadin (CB), and the producer is a pottery art organism.

Experimental method

1. Obtaining and culturing thyroid undifferentiated carcinoma cell lines.

8505C was obtained from the German collection of microorganisms and cell cultures (DSMZ), KHM-5M, CAL62 was purchased from the China cell line resource pool (Shanghai, china). All cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin, in 37% incubator containing 5% CO 2.

Cck8 cell viability assay to detect cytotoxicity of CB, S63845 in thyroid undifferentiated carcinoma cell lines.

Thyroid undifferentiated carcinoma cells were plated in 96-well plates (5000 cells per well) and then incubated in a 5% CO2 incubator at 37 ℃ for 24 hours prior to treatment. Cells 24h were then treated with different concentrations of CB, S63845. 10 mu l of CCK-8 and 90 mu l of CCK-8 are respectively added into each hole of the test pointsRPMI 1640 medium with 10% FBS, then at 37℃with 5% CO ₂ Cultures were grown down to approximately 2h, and then OD (optical density) was detected at a wavelength of 450nm using a Synergy LX Multi-Mode Reader (BioTek Instruments, USA).

CB is molecular docking with MCL-1.

And (5) docking the key active ingredients with the core target by using PyMOL, autoDockTool 1.5.6 software. The structural files (mol 2 format) of the key active ingredients and the protein crystal structures (PDB format) of the core targets are downloaded and obtained from the PubCHem and RSCB PDB databases respectively.

4. Determination of mortality of thyroid undifferentiated carcinoma cells.

Cell death was detected using an Annexin V-FITC and PI kit (Liankebio, china) according to the manufacturer's instructions. ATC cells (1.8x10) ⁵ Cells/well) were seeded in 6-well plates and treated with different drugs after complete attachment. After incubation at 37 ℃ for 24h, adherent and floating cells were taken, washed 2 times with ice-cold PBS, and then suspended with 1 x binding buffer. Cells were labeled with 1 μl Annexin V-FITC and 2 μl PI and incubated for 5min at room temperature. Apoptosis was detected by flow cytometry and cell death was determined by Flowjo. Cell death was qualitatively detected using a live/dead cell staining kit (Calcein AM PI).

5. Thyroid undifferentiated carcinoma cell migration and invasiveness test.

To detect changes in tumor cell migration, invasive capacity, a transwell cell culture chamber (Corning Costar Corp, cambridge, MA, USA) was used. The upper chamber surface of the bottom membrane of the Transwell chamber was coated with 50mg/L Matrigel ™ (BD Biocoat) 1:4 dilution 1 hr before detection of changes in invasive capacity and incubated at 37 ℃. ATC cells (migration: 2.5x10) ⁴ A cell; attack: 8X 10 ⁴ Cells) were inoculated into the upper chamber with serum-free medium and the lower chamber was filled with 800ml of 10% FBS medium containing CB drugs at different concentrations. The 24-well plate was then placed in a humid incubator, incubated at 37 ℃ for 24 hours with 5% CO2, and challenged for 48 hours. Cells that did not pass through the filter were rubbed off with a cotton swab. Migrated cells on the back of the filter were fixed with 4% PFA (Sigma) for 30 min0.01% crystal violet was stained for 30 minutes, photographed under an optical microscope and quantified. And quantified with ImageJ software (https:// ImageJ. Nih. Gov/ij /). All experiments were repeated three times. Detection of colony formation.

6. In vivo nude mice transplantation tumor experiments.

Female nude mice (BALB/c, 16-20 g, 3-4 weeks old) were purchased from Shanghai Schlenk Biotechnology, kept in the laboratory animal center of the national hospital in Zhejiang province, china, cycled for 12 hours around the clock, and provided food and water free. Mice were subcutaneously injected with a single 8505C cell suspension (4X 10) ⁶ Cell/100 μl). After 1 week of transplantation, mice were randomly divided into different groups (n=6/group) and each were intraperitoneally injected daily with 5% dmso+40% peg300+10% tween80 and 45% ddH2O, 100 μl/mouse, CB (2.5, 5 mg/kg) in 5% dmso+40% peg300+10% tween80 and 45% ddH2O, for 14 consecutive days. Tumor volumes and body weights were recorded every other day. The xenograft tumor volume (mm 3) was calculated as 0.5× (shortest diameter) 2× (longest diameter). At the end of the experiment, all mice were sacrificed and the dissected xenograft tissues were weighed. All animal experiments were performed according to the protocol approved by the animal ethics committee (IACUC-a 20220051) of the people hospital, zhejiang province.

7. Western blotting experiments.

Cells were collected 24h after drug treatment and were dissolved in PMSF-containing western and IP lysis buffer (P0013, institute of biotechnology, china bi) for 10 min. The total protein concentration was quantitatively determined using BCA (bisdiphenolic acid) protein assay kit (sammer feier science company, usa). Protein samples were resolved on SDS-PAGE preformed Tris-Gly gel (4-20%, #P0524M, beyotime Biotechnology institute, china) and transferred to PVDF membrane. Membranes were blocked with TBST of 1% Tween-20 containing 5% skim milk for 1 hour. Subsequently, the membranes were incubated with the corresponding primary antibodies overnight at 4 ℃, then incubated with the appropriate secondary antibodies coupled to HRP for 60 minutes at room temperature. The membranes were analyzed with the fdio-dura ECL kit (#fd 8020, fdio Science, china) and imaged with a ChemiDoc-MP imager (Bio-Rad, usa). Band densities were quantified with ImageJ software.

Experimental results

Toxicity of cb and S63845 to cells.

The CCK8 is adopted to detect the proliferation conditions of ATC cells 8505C, KHM-5M, C643 and CAL-62 after treatment for 24 hours, and the proliferation conditions of 8505C cells after treatment for 24 hours are S63845, so that the results show that CB and S63845 can induce the dose-dependent death of ATC cells, and the same dose of CB has better curative effect. In 8505C, CB reached 50% of the killer cells at a drug concentration of 50nM, while S63845 reached 50% of the killer cells at a drug concentration of about 10000 nM.

As shown in fig. 1, IC50 s of CB in thyroid undifferentiated carcinoma cells are shown as follows: 8505C:52.23nM; KHM-5M:218.5 nM; CAL-62:158.0 And nM. The results indicated that CB has the best potency in 8505C and that CAL-62 has relatively poor potency in KHM-5M. The effect of the existing inhibitors was subsequently further verified with MCL-1 high potency inhibitor-S63845 in 8505C cells. Higher doses of S63845 induce the same cell death, 4000nM CB can cause more than 90% ATC cell death, while 4000nM S63845 can cause less than about 10% ATC cell death. CB is therefore expected to replace S63845 as a novel MCL-1 inhibitor.

2. CB interfaces with molecules of MCL-1.

As shown in FIG. 2A, the molecular docking of the MCL-1 protein and CB in the 3D structure is verified, and the fact that the binding energy of the CB and the MCL-1 is less than or equal to-8.0 kcal/mol shows that the binding activity is stronger. This is also true in the 2D structure shown in fig. 2B.

3.CB significantly promoted 8505c cell death by cell flow detection, with CB at the same dose as S63845.

As shown in FIG. 3, at the same doses of CB, S63845 (0 nM,100nM,200nM,400 nM), the survival rates of CB on 8505C cells were about 95%,70%,50%,40%, respectively; the survival rate of KHM-5M is about 97%,93%,90% and 83%, and that of CAL-62 is about 96%,94%,93% and 92%, respectively; the survival rate of S63845 was 98%,97%,97% and 95% when applied to 8505C cells. I.e. the same dose of CB may specifically lead to more 8505C deaths.

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

Inoculating 2×10 with 24-well plate ⁵ After 24h on cells/well 8505C, CB5 treatment was given at different concentrations for 24h, while a control group was set. The treated 8505C cells were then stained for Calcein-AM and PI using IF techniques, which showed that 8505C cells had dose-dependent cell death. And WB can detect dose-dependent Mcl-1 decrease, PARP cleavage and cytoC increase, namely CB can down regulate MCL-1 protein expression in ATC cells so as to promote tumor cell apoptosis. The apoptosis marker index PARP is cleaved, and cytoC is also detected to be increased, and CB is also proved to induce apoptosis.

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

Transwell laboratory experiments as shown in FIG. 5 show that CB can dose-dependently inhibit migration and invasion of 8505C cells.

6. In animal experiments, CB groups (2.5 mg/Kg, 5 mg/Kg) of different concentrations were set, and the treatment effect on thyroid undifferentiated carcinoma in mice was set. Both low and high doses of CB can significantly inhibit tumor growth and high doses of CB can be more effective the higher the dose (fig. 6a,6 c). The CB low and high dose groups had significantly reduced tumor weights and tumor volumes relative to NC groups (fig. 6D, 6E) with no significant change in body weight in mice (fig. 6B).

7. As shown in fig. 7, the heart, liver, spleen and kidney HE staining of animal tissues of different dose groups is not different, i.e. the result shows that CB of the dose has no toxic or side effect 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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