CN110585433A

CN110585433A - Application of BRAF-V600E inhibitor and drug for treating thyroid cancer

Info

Publication number: CN110585433A
Application number: CN201910903458.0A
Authority: CN
Inventors: 高岩岩; 李培峰
Original assignee: Qingdao University
Current assignee: Qingdao University
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2019-12-20

Abstract

The invention provides an application of an inhibitor of BRAF-V600E and a medicine for treating thyroid cancer, and relates to the technical field of biology. According to the invention, the BRAF-V600E inhibitor is applied to the preparation of the medicine for treating thyroid cancer, and the treatment is carried out aiming at the target spot, so that the sensitivity of thyroid cancer radiotherapy can be effectively enhanced, and the effect of treating thyroid cancer is greatly improved. The medicine for treating thyroid cancer provided by the invention comprises an inhibitor of BRAF-V600E. The active ingredient of the medicine is an inhibitor of BRAF-V600E, so that the sensitivity of thyroid cancer radiotherapy can be effectively enhanced through targeted therapy, the effect of treating thyroid cancer is greatly improved, cells which are not subjected to tumor transformation are not affected, and the medicine is safe, non-toxic and small in side effect.

Description

Application of BRAF-V600E inhibitor and drug for treating thyroid cancer

Technical Field

The invention relates to the technical field of biology, in particular to application of a BRAF-V600E inhibitor and a medicine for treating thyroid cancer.

Background

Thyroid cancer is the most common endocrine malignancy. BRAF gene mutations are the most prominent type of gene mutations in Papillary Thyroid Carcinoma (PTC). Mutation of BRAF gene usually occurs from T to a at 1799, i.e. from valine to glutamic acid at position 600 of the protein sequence (V600E). In recent years, the incidence of thyroid cancer in China is increasing year by year. Therefore, intensive research is urgently needed on the pathology and molecular mechanism of thyroid cancer.

Treatment of thyroid cancer is usually by surgical resection or in combination with post-operative radioiodine 131 treatment. The radioactive iodine 131 treatment is that thyroid cells have special affinity to iodide, after a certain amount of iodine 131 is taken orally, the iodine can be absorbed by the thyroid gland in a large amount, and beta rays emitted by radioactive iodine can selectively destroy thyroid gland acinar epithelium without influencing adjacent tissues, so that the effect of specifically killing thyroid gland tissues is achieved. The radioactive iodine therapy is to destroy the cell chromosome DNA by the ionizing radiation generated by the decay of iodine 131, so that the cell necrosis caused by DNA damage is generated, thereby achieving the purpose of killing the tumor cells. The most serious consequence of ionizing radiation is DNA double-strand breaks (DSBs) that, if not successfully repaired, cells cannot divide but die by apoptosis, etc. Thus, there is a necessary link between apoptotic mechanisms and susceptibility to radioiodine 131 treatment. Malignant tumor cells are resistant to radiation therapy, associated with their ability to escape apoptosis. Therefore, the research on the mechanism of cancer cell escape from apoptosis has instructive significance for cancer radiotherapy and radioiodine treatment of thyroid cancer.

The mitochondrial apoptotic pathway is one of the major pathways of apoptosis. While mitochondral permeability transition pore (mPTPs) are non-selective porous channels located on the inner mitochondrial membrane, the closed state of mPTP is thought to play a very important regulatory role in the apoptotic process. mPTP promotes apoptosis of cells, so it plays a very important role in carcinogenesis. Cancer cells inhibit this pore opening by reducing sensitivity of mPTPs to escape apoptosis. The regulatory mechanism of mPTP in cancer cells, including Ca in mitochondria²⁺Overload, ROS, etc.

At present, some iodine-refractory thyroid cancer patients exist clinically, are insensitive to radioactive iodine treatment, and cannot kill cancer cells by using iodine 131 with a conventional dose. The patients can be treated only by repeatedly taking or increasing the dose of the iodine 131, but the treatment effect is not ideal, and radiation side effects, such as pneumonia or pulmonary fibrosis, salivary gland injury, secondary tumor mild increase and the like, can be generated.

Therefore, how to improve the radiation sensitivity of iodine 131 of the thyroid patients with iodine refractory is a problem to be solved urgently.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide application of a BRAF-V600E inhibitor in preparation of a medicine for treating thyroid cancer, so as to solve the technical problems that thyroid cancer patients are insensitive to radioactive iodine treatment, cannot kill cancer cells by using iodine 131 with a conventional dose and cannot treat thyroid cancer safely and effectively in the prior art.

The invention provides an application of an inhibitor of BRAF-V600E in preparing a medicament for treating thyroid cancer.

Further, the BRAF-V600E is BRAF-V600E positioned on mitochondria;

preferably, the BRAF-V600E positioned on mitochondria has an amino acid sequence shown as SEQ ID NO. 1.

Further, the inhibitor of BRAF-V600E is an agent for inhibiting the localization of BRAF-V600E on mitochondria;

preferably, the inhibitor of BRAF-V600E comprises one or more of Vemurafenib, Dabrafinib, or PLX-8394.

Further, the inhibitor of BRAF-V600E treats thyroid cancer by enhancing the sensitivity of radioiodine therapy.

Further, the inhibitors of BRAF-V600E enhance the sensitivity of radioiodine therapy by promoting apoptosis.

Further, the inhibitor of BRAF-V600E promotes apoptosis by opening mitochondrial mPTP pores.

Further, the BRAF-V600E inhibitor can open the mitochondrial mPTP channel by promoting the phosphorylation of CyP-D.

Further, the BRAF-V600E inhibitor promotes the phosphorylation of CyP-D by enhancing the activity of GSK3 β.

Further, the BRAF-V600E inhibitor enhances the activity of GSK3 β by inhibiting phosphorylation of Ser9 of GSK3 β downstream of ERK in mitochondria.

Preferably, the inhibitor of BRAF-V600E inhibits phosphorylation of Ser9 of GSK3 β downstream of ERK in mitochondria by inhibiting ERK activity in mitochondria.

In addition, the invention also provides a medicament for treating thyroid cancer, which comprises an inhibitor of BRAF-V600E;

preferably, the BRAF-V600E is BRAF-V600E positioned on mitochondria;

preferably, the BRAF-V600E positioned on mitochondria has an amino acid sequence shown as SEQ ID NO. 1;

preferably, the inhibitor of BRAF-V600E is an agent for inhibiting the localization of BRAF-V600E on mitochondria;

A large number of experiments prove that BRAF-V600E can resist apoptosis and reduce the sensitivity of thyroid cancer to radioactive iodine, which is an important path for resisting apoptosis of BRAF-V600E mutant thyroid cancer. The conventional dose of iodine 131 cannot kill cancer cells through the pathway, so that the thyroid cancer cannot be treated with traditional postoperative radioactive iodine 131, and the BRAF-V600E anti-apoptosis pathway provides an important target for radioactive iodine treatment of the thyroid cancer. According to the invention, the BRAF-V600E inhibitor is applied to the preparation of the medicine for treating thyroid cancer, and the treatment is carried out aiming at the target spot, so that the sensitivity of thyroid cancer radiotherapy can be effectively enhanced, and the effect of treating thyroid cancer is greatly improved. In addition, the BRAF-V600E inhibitor has no influence on cells without tumor transformation on the basis of targeting anticancer, and is safe, nontoxic and small in side effect.

The medicine for treating thyroid cancer provided by the invention comprises an inhibitor of BRAF-V600E. The active ingredient of the medicine is an inhibitor of BRAF-V600E, so that the sensitivity of thyroid cancer radiotherapy can be effectively enhanced through targeted therapy, the effect of treating thyroid cancer is greatly improved, cells which are not subjected to tumor transformation are not affected, and the medicine is safe, non-toxic and small in side effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a diagram showing the results of the detection of various indexes of the constructed cells transiently overexpressed in BRAF WT and BRAF-V600E according to example 1 of the present invention;

FIG. 2 is a graph showing the results of Western blot detection of the phosphorylation level of ERK in mitochondria of cells overexpressing BRAF-V600E according to example 2 of the present invention;

FIG. 3 is a graph showing the result of determining the ERK phosphorylation level of mitochondrial localization of thyroid cancer cells by Western blot, provided in example 3 of the present invention;

FIG. 4 is a graph showing the results of Western blot measurement of the p-ERK/ERK levels of sorafenib-treated thyroid cancer cells as provided in example 4 of the present invention;

FIG. 5 is a graph of the results of Western blot determination of the level of mitochondrial localization p-ERK/ERK of sofafenib-treated BRAF-overexpressing cells provided in example 5 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In a preferred embodiment, the BRAF-V600E is BRAF-V600E localized on mitochondria.

Compared with the cells over-expressed by BRAF WT, the cells over-expressed by BRAF-V600E can resist apoptosis induced by an apoptosis inducer STS and the like, so that the mitochondria localization of BRAF-V600E can mediate the anti-apoptosis effect of the cells, thereby inhibiting the treatment of thyroid cancer. Therefore, the inhibitor aiming at BRAF-V600E positioned on mitochondria can be used for solving the problem that BRAF-V600E resists apoptosis inducers.

Preferably, the BRAF-V600E located on mitochondria has an amino acid sequence shown as SEQ ID NO. 1.

In a preferred embodiment, the inhibitor of BRAF-V600E is an agent that inhibits the localization of BRAF-V600E on mitochondria.

The invention discovers that the conventional BRAF-V600E inhibitor, such as sorafenib and MEK inhibitor U0126 and the BRAF specific inhibitor PLX4720, can not change the mitochondrial localization and the anti-apoptotic activity of BRAF-V600E in thyroid cancer. According to the invention, the specific reagent for inhibiting the BRAF-V600E from being positioned on mitochondria is used as the inhibitor of BRAF-V600E, and the BRAF-V600E positioned on mitochondria can be used as a target gene to quickly and accurately inhibit the activity of the gene, so that the aim of treating thyroid cancer is fulfilled.

In a preferred embodiment, an inhibitor of BRAF-V600E treats thyroid cancer by enhancing the sensitivity of radioiodine therapy.

The V600E mutation is commonly generated in BRAF in the recurrent thyroid cancer of the iodine-refractory thyroid cancer patient, which shows that the thyroid cancer patient carrying the BRAF-V600E mutation has reduced sensitivity to iodine 131 treatment, tumor cells are easy to recur, and lymph node or distant metastasis is generated. In addition, BRAF-V600E also affects the expression of thyroid cell iodometabolism related genes including Thyroid Peroxisome (TPO), thyroid hormone receptor (TSHR), thyroglobulin (Tg) and the like, and further affects the uptake of radioactive iodine by thyroid cells and the retention of radioactive iodine in the thyroid cells. The inhibitor of BRAF-V600E applied by the invention can be used for treating thyroid cancer by enhancing the sensitivity of radioiodine treatment.

In a preferred embodiment, an inhibitor of BRAF-V600E enhances the sensitivity of radioiodine therapy by promoting apoptosis.

In a preferred embodiment, an inhibitor of BRAF-V600E promotes apoptosis by opening mitochondrial mPTP pore channels.

The research of the invention finds that the mitochondria locates BRAF-V600E, and reduces the sensitivity of thyroid cancer to radioactive iodine by inhibiting the opening anti-apoptosis effect of mitochondria mTP. The inhibitor of BRAF-V600E can effectively reduce the activity of BRAF-V600E positioned by mitochondria, thereby opening the mPTP pore passage of mitochondria, promoting apoptosis, enhancing the sensitivity of radioactive iodine treatment and achieving the aim of treating thyroid cancer.

In a preferred embodiment, an inhibitor of BRAF-V600E opens mitochondrial mPTP pathways by promoting phosphorylation of CyP-D.

In a preferred embodiment, the inhibitor of BRAF-V600E promotes the phosphorylation of CyP-D by enhancing the activity of GSK3 β.

In a preferred embodiment, the inhibitor of BRAF-V600E enhances the activity of GSK3 β by inhibiting phosphorylation of Ser9 of GSK3 β downstream of ERK in the mitochondria.

Due to the high kinase activity of BRAF-V600E, the phosphorylation level of ERK in mitochondria of a BRAF-V600E overexpressed cell is obviously higher than that of a BRAF WT overexpressed cell, which indicates that the mitochondria-localized BRAF-V600E in thyroid cancer causes the over-activation of ERK in mitochondria, and the mitochondria-localized ERK promotes the phosphorylation of Ser9 of downstream GSK3 beta to inhibit the activity of GSK3 beta, so that the phosphorylation of downstream CyP-D is inhibited, the CyP-D is not combined with the mitochondrial mTP pore channel, PTP is in a closed state, and the apoptosis is inhibited, which is an important path for resisting the apoptosis of the BRAF-V600E mutated thyroid cancer. Mitochondrial localization of BRAF-V600E against apoptosis through this pathway reduces the sensitivity of thyroid cancer to radioiodine therapy. Therefore, mitochondrially localized BRAF-V600E anti-apoptotic pathway provides an important target for radioiodine therapy of thyroid cancer.

In addition, the invention also provides a medicine for treating thyroid cancer, which comprises an inhibitor of BRAF-V600E.

The active ingredient of the drug for treating thyroid cancer provided by the invention is an inhibitor of BRAF-V600E, so that the sensitivity of thyroid cancer radiotherapy can be effectively enhanced through targeted therapy, the effect of treating thyroid cancer is greatly improved, cells which are not subjected to tumor transformation are not affected, and the drug is safe, non-toxic and small in side effect.

To facilitate a clearer understanding of the contents of the present invention, reference will now be made in detail to the following specific embodiments.

EXAMPLE 1 construction of cells transiently overexpressing BRAF WT and BRAF-V600E

To study the effect of BRAF WT (BRAF wild type) and BRAF-V600E (BRAF mutant) proteins on mitochondrial function. In this example, HEK293T cells were selected, and overexpression plasmids including pCMV-N-Flag, pCMV-N-Flag-BRAF WT and pCMV-N-Flag-BRAF-V600E were transfected into HEK293T cells for overexpression using a lipofection method, and the overexpression effect of BRAF was determined using a Western blotting method. The results are shown in fig. 1, indicating that BRAF is expressed in increased amounts in cells and that the level of phosphorylated ERK is enhanced.

The specific operation of this embodiment is as follows:

construction of overexpression plasmids: the BRAF sequences of BRAF WT and BRAF T1799A site mutation are constructed on a pCMV-N-Flag vector. The vector is kana resistant.

And (3) carrying out lipofection: liposome Lipofetamine-3000(Thermo scientific) transfects HEK293T cells, and the ratio of plasmid to Lipofetamine-3000 is 2: 1, fluid exchange was performed 6 hours after transfection, and cells were collected 24 hours later.

Protein immunoblotting:

liposome was transfected into HEK293T cells for 24 hours, RIPA lysate was added, repeated freeze-thawing was carried out three times at-80 ℃, centrifugation was carried out at 12,000rpm for 15 minutes, and the supernatant was aspirated to determine the protein concentration.

Preparing 15% SDS-PAGE gel, adding a certain volume of loading buffer solution to a sample with the measured protein concentration, heating at 95 ℃ for 10 minutes, taking 50 mu g of protein, adding the protein into the 15% SDS-PAGE gel, running to the separation gel at a constant pressure of 80V, and then running for about 3 hours.

The protein samples on the gel were transferred to PVDF membrane, blocked with 5% skim milk for 1 hour, and then incubated overnight with anti-Flag antibody (sigma), p-ERK antibody (CST), and Tubulin antibody (CST) diluted at 1: 1000. The membranes were washed 3 times for 10 minutes each with TBST. A1: 5000 dilution of horseradish peroxidase-labeled secondary rabbit antibody (santa cruz) was added and incubated for 1 hour. TBST (150mM NaCl, 20mM Tris-HCl, pH 7.4, Tween-200.05%) was washed three times, then added with hypersensitivity ECL chemiluminescence reagent (Biyutian Corp.), and exposed in a dark box.

Western blot results show that BRAF WT or BRAF-V600E is over-expressed in HEK293T cells, and a Western blot method is used for determining the ratio of p-ERK/ERK. Significant increase in p-ERK/ERK was determined in HEK293T over-expressed in BRAF-V600E compared to cells over-expressed in BRAF WT.

Example 2Western blot detection of the phosphorylation level of ERK in mitochondria of cells overexpressing BRAF-V600E

In order to study the phosphorylation level of BRAF-V600E on ERK localized by mitochondria, the example separates the cytoplasm and the mitochondrial components of BRAF overexpression cells, and Western blot measures the level of p-ERK/ERK localized by mitochondria, and the result is shown in figure 2, which shows that the overexpression of BRAF-V600E promotes the level of p-ERK/ERK localized by mitochondria.

The specific operation of this embodiment is as follows:

HEK293T cells pCMV-N-Flag, pCMV-N-Flag-BRAF WT and pCMV-N-Flag-BRAF-V600E are transfected by liposome, so that the BRAF WT and BRAF-V600E proteins are over-expressed in the cells (the specific experimental steps refer to the above).

After 24 hours of transfection, the cytoplasm and mitochondria of the cells were separated by a mitochondrial isolation kit (Thermo scientific), according to the kit instructions. After the mitochondria obtained by the isolation were lysed with a mitochondrial lysate, the cell was centrifuged at 12,000rpm for 10 minutes, and the supernatant was collected and the protein concentration was measured by the BCA method.

After quantification of cytoplasmic and mitochondrial protein components, 15% SDS-PAGE electrophoresis and western blot were performed, the procedure was as described above, with Tubulin and COX IV indicating the cytoplasm and mitochondria, respectively, and incubated overnight with anti-Flag antibody, Tubulin antibody, COX IV antibody and p-ERK antibody, the next day, 1:5000 dilution of horseradish peroxidase-labeled rabbit secondary antibody or mouse secondary antibody (santa cruz).

Western blot results show that BRAF WT and BRAF-V600E are over-expressed in cells, mitochondria and cytoplasm are successfully separated, and mitochondria are basically free from cytoplasmic pollution and cytoplasm are free from mitochondrial pollution. In addition, in addition to the obvious increase of p-ERK/ERK in cytoplasm of the cells over-expressed in BRAF-V600E, p-ERK/ERK is also obviously enhanced in mitochondria of the cells over-expressed in BRAF V600. This indicates that the mitochondrial localization of BRAF-V600E promotes p-ERK/ERK levels in mitochondria.

Example 3Western blot determination of ERK phosphorylation levels in mitochondrial localization of thyroid carcinoma cells

To investigate the effect of endogenous BRAF on mitochondrially localized ERK phosphorylation levels, this example analyzed mitochondrially localized ERK phosphorylation levels in various thyroid cells including BRAF-V600E mutant thyroid cancer cells (BCPAP, 8505C), BRAF WT thyroid cancer cells (FTC-133), and normal thyroid cells (nth-ori-3.1). Mitochondrial proteins are extracted after cell culture and are subjected to western blot analysis, and the levels of ERK and p-ERK on mitochondria are measured. The results are shown in FIG. 3, which shows that the phosphorylation level of ERK localized on mitochondria of BRAF-V600E mutant thyroid cancer cells (BCPAP, 8505C) is higher than that of BRAF WT thyroid cancer cells (FTC-133) and normal thyroid cells (Nthy-ori-3.1). This result demonstrates that the BRAF-V600E mutant cancer cell ERK is constitutively activated at mitochondria.

The specific operation of this embodiment is as follows:

thyroid cells were cultured in 10cm dishes, with BCPAP cells cultured with RIPM 1640+ 10% newborn bovine serum, 8505C with DMEM: f12(1:1) + 10% newborn bovine serum, Nthy-ori-3.1 with RIPM 1640+ 10% fetal bovine serum, FTC-133 cells with DMEM high sugar medium + 10% newborn bovine serum culture.

After the cells were confluent, the cells were scraped off, centrifuged at 12,000rpm, and the cytoplasm and mitochondria of the cells were separated using a mitochondrial isolation kit (Thermo scientific), in which mitochondrial lysates were used to lyse mitochondria and cytoplasmic and mitochondrial proteins were quantified using the BCA method.

Detection was carried out by 15% SDS-PAGE and Western blot method using anti-BRAF antibody, COX IV antibody and p-ERK, ERK antibody overnight, and the next day, using horseradish peroxidase-labeled rabbit secondary antibody or mouse secondary antibody (santa cruz) diluted 1:5000 as described above.

Western blot results show that the phosphorylation level of ERK localized on mitochondria of BRAF-V600E mutant thyroid cancer cells (BCPAP, 8505C) is higher than that of BRAF WT thyroid cancer cells (FTC-133) and normal thyroid cells (Nthy-ori-3.1). This suggests that the BRAF-V600E mutant cancer cell ERK is constitutively activated at mitochondria.

Example 4Western blot assay for the level of p-ERK/ERK in sorafenib-treated thyroid carcinoma cells

In the embodiment, HEK293T cells transiently overexpressing BRAF WT and BRAF-V600E are treated by 10 mu M sorafenib for 24 hours, and protein is extracted to measure the level of p-ERK, so that the sorafenib is found to effectively inhibit p-ERK/ERK after 24 hours of treatment.

The specific operation of this embodiment is as follows:

three plasmids, pCMV-N-Flag-BRAF WT and pCMV-N-Flag-BRAF-V600E, were transiently transfected into HEK293T cells and then treated with 10. mu.M sorafenib for 24 hours. The RIPA lysate is used for cell lysis, protein is extracted, and the protein concentration is determined by a BCA method.

15% SDS-PAGE and Western blot analysis were performed and the levels of p-ERK/ERK were determined using antibodies to p-ERK and ERK. The results are shown in FIG. 4, and it was found that 10. mu.M sorafenib significantly inhibited the level of p-ERK/ERK in the over-expressed BRAF cells.

Example 5Western blot assay for the level of mitochondrial localization of p-ERK/ERK in sofafenib-treated BRAF-overexpressing cells

In the embodiment, HEK293T cells transiently overexpressing BRAF WT and BRAF-V600E are treated by 10 mu M sorafenib for 24 hours, so that the level of p-ERK/ERK can be obviously inhibited. Meanwhile, we extracted mitochondria of cells, and western blot analysis of phosphorylation levels of ERK at mitochondrial localization of sorafenib-treated and control groups, and the results are shown in fig. 5, which shows that the mitochondrial p-ERK level of HEK293T cells of BRAF-V600E treated by sorafenib is significantly higher than that of HEK293T cells of BRAF WT, and BRAF WT and BRAF-V600E are still localized on mitochondria. This suggests that although sorafenib inhibits the level of p-ERK in the cell as a whole, sorafenib cannot inhibit the mitochondrial localization of BRAF WT and BRAF-V600E, and that the mitochondrial localization of BRAF-V600E regulates ERK phosphorylation in mitochondria such that its enhanced ERK phosphorylation level is not reversed by sorafenib.

The specific operation of this embodiment is as follows:

three plasmids, pCMV-N-Flag-BRAF WT and pCMV-N-Flag-BRAF-V600E, were transiently transfected into HEK293T cells and then treated with 10. mu.M sorafenib for 24 hours. The cytoplasm and mitochondria of the cells were separated using a mitochondrial isolation kit (Thermo scientific), and the mitochondria were lysed with a mitochondrial lysate and the cytosol and mitochondrial proteins were quantified by the BCA method as described above.

Mitochondrial proteins were detected by 15% SDS-PAGE and Western blot methods described above, with COX IV indicating mitochondria and Flag indicating overexpression of BRAF.

Western blot results show that sorafenib cannot inhibit the mitochondrial localization of BRAF WT and BRAF-V600E, and the BRAF-V600E with mitochondrial localization regulates the phosphorylation of ERK in mitochondria, so that the enhanced phosphorylation level of ERK is not reversed by sorafenib.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Qingdao university

Application of <120> BRAF-V600E inhibitor and drug for treating thyroid cancer

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<170> PatentIn version 3.5

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Claims

Use of an inhibitor of BRAF-V600E in the manufacture of a medicament for the treatment of thyroid cancer.
2. The use according to claim 1, wherein said BRAF-V600E is BRAF-V600E localized on mitochondria;

preferably, the BRAF-V600E positioned on mitochondria has an amino acid sequence shown as SEQ ID NO. 1.
3. The use according to claim 1, wherein the inhibitor of BRAF-V600E is an agent that inhibits the mitochondrial localization of BRAF-V600E;

preferably, the inhibitor of BRAF-V600E comprises one or more of Vemurafenib, Dabrafinib, or PLX-8394.
4. The use according to claim 1, wherein the inhibitor of BRAF-V600E is used to treat thyroid cancer by enhancing the sensitivity of radioiodine therapy.
5. The use of claim 4, wherein the inhibitor of BRAF-V600E enhances the sensitivity of radioiodine therapy by promoting apoptosis.
6. The use of claim 5, wherein the inhibitor of BRAF-V600E promotes apoptosis by opening mitochondrial mPTP pore channels.
7. The use of claim 6, wherein the inhibitor of BRAF-V600E opens mitochondrial mPTP pathways by promoting phosphorylation of CyP-D.
8. The use according to claim 7, wherein the inhibitor of BRAF-V600E promotes the phosphorylation of CyP-D by enhancing the activity of GSK3 β.
9. The use according to claim 8, wherein the inhibitor of BRAF-V600E enhances the activity of GSK3 β by inhibiting phosphorylation of Ser9 of GSK3 β downstream of ERK in mitochondria;

preferably, the inhibitor of BRAF-V600E inhibits phosphorylation of Ser9 of GSK3 β downstream of ERK in mitochondria by inhibiting ERK activity in mitochondria.
10. A medicament for the treatment of thyroid cancer, comprising an inhibitor of BRAF-V600E;

preferably, the BRAF-V600E is BRAF-V600E positioned on mitochondria;

preferably, the BRAF-V600E positioned on mitochondria has an amino acid sequence shown as SEQ ID NO. 1;

preferably, the inhibitor of BRAF-V600E is an agent for inhibiting the localization of BRAF-V600E on mitochondria;

preferably, the inhibitor of BRAF-V600E comprises one or more of Vemurafenib, Dabrafinib, or PLX-8394.