CN113018301A

CN113018301A - Application of JQ1 and Erlotinib composition in preparation of liver cancer treatment drugs and liver cancer treatment drugs

Info

Publication number: CN113018301A
Application number: CN201911250020.3A
Authority: CN
Inventors: 刘扬; 尹雅蕾
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2019-12-09
Filing date: 2019-12-09
Publication date: 2021-06-25

Abstract

The invention relates to an application of a composition of a BET bromopain inhibitor and an EGFR inhibitor in treating liver cancer. The BET bromodomain inhibitor JQ1 and the EGFR inhibitor Erlotinib are mixed for use, so that the effects of inhibiting proliferation, clone formation, transfer and the like of liver cancer cells can be effectively achieved. The combined use of the two has obviously improved inhibition effect on the growth of the liver cancer tumor, and can be used for treating the EGFR mutant liver cancer.

Description

Application of JQ1 and Erlotinib composition in preparation of liver cancer treatment drugs and liver cancer treatment drugs

Technical Field

The invention only relates to a method for treating liver cancer carrying EGFR mutation by using a composition of a BET bromopain inhibitor JQ1 and an EGFR inhibitor Erlotinib described in the application, and belongs to the technical field of cell biology and medical treatment.

Background

In recent years, liver cancer has become one of the most common malignant tumors threatening human health, with the incidence being at the 6 th position of cancer onset and the mortality at the 3 rd position. In China, liver cancer has high morbidity and mortality, and liver cancer patients account for 55% of patients all over the world, so that the health and life of people are seriously affected.

A great deal of research shows that the occurrence and development of tumors are closely related to the mutation and expression of genes. The pathogenic factors of the tumor can cause the cell to become cancerous by damaging cellular DNA, inducing the cellular gene to generate mutation and causing the expression disorder of the cellular gene, and changing the cellular protein and metabolism level. The occurrence of liver cancer is a complex process, and external or internal factors cause the genetic change of liver cells, so that the growth and proliferation of the cells are abnormal, and tumors are formed. Research has shown that the occurrence and development of liver cancer are associated with genetic changes in various oncogenes and cancer suppressor genes. Among them, the related oncogenes related to the development of liver cancer mainly include ras, c-myc, c-erbB-2, c-fos, bcl-2, p53, p16, etc. In addition, abnormal activation of signaling pathways related to tumorigenesis and development, such as EGFR-MAPK-ERK, PI3K-AKT, Wnt/beta-catenin signaling pathways and the like, can be closely related to liver cancer.

MYC is a nucleoprotein transcription factor in oncogenes, is a key factor for regulating various growth signal pathways, has mutation or overexpression in 70 percent of tumors, and is closely related to the occurrence and development of cancers. MYC and Max protein form heterodimer, and then the heterodimer is specifically recognized and combined to a gene promoter region to regulate gene transcription. MYC regulates the transcription of a plurality of protein genes, including the gene transcription of related proteins of cell cycle, growth, metabolism and the like, and plays an important regulating and controlling role in various aspects of cell proliferation, differentiation, transfer and the like. Thus, MYC protein overexpression causes rapid cell proliferation, ultimately leading to cellular abnormalities and carcinogenesis. The occurrence and development of liver cancer are closely related to the expression of MYC, which is a key regulatory factor in the malignant transformation process of liver cancer, and researches show that liver tissue-specific MYC transgenic mice can directly cause the occurrence of liver cancer (78, 79). Analysis of the TCGA database showed that the amplification of the liver cancer MYC gene reached more than 20% (fig. 1). Clinical studies found that the expression level of MYC protein in liver cancer tissues is remarkably higher (P <0.05) than that in liver tissues beside cancer. In addition, MYC gene amplification is negatively correlated with recurrence and overall survival for patients after liver cancer surgery (77). Therefore, MYC and the regulation mechanism thereof become main targets of targeted therapy of liver cancer. BET bromodomain inhibitors can inhibit tumor growth by blocking MYC protein expression and have been reported for use in a variety of tumor therapy studies. However, the application of JQ1 to liver cancer treatment is rarely reported.

In tumor cells, a variety of signaling pathways are possible to regulate MYC protein levels in cells. Such as PI3K-AKT, MAPK-ERK signaling pathways, etc., can regulate MYC protein levels through both transcriptional and post-translational modifications. The MAPK-ERK signal pathway enters a nucleus after being activated by growth factors or cytokines, expression and phosphorylation of transcription factor MYC in the nucleus can be regulated, and in addition, p-ERK can phosphorylate FBW7 to promote self-ubiquitination degradation of the FBW7, so that ubiquitination degradation of MYC protein is inhibited. Therefore, activation of the MAPK-ERK signaling pathway may have important regulatory effects on resistance of JQ1 in cells.

The factors contributing to the resistance of targeted therapies are mainly due to mutations or changes in expression of target genes. Among them, mutations in the EGFR gene are a major cause of drug resistance. The phosphorylated EGFR can be combined with a growth factor receptor binding protein Grb2 complex to activate Ras-Raf-MAPK signals, so that the biological functions of ERK phosphorylation, cell proliferation, differentiation, invasion, apoptosis and the like are enhanced.

Therefore, the combined use of the MYC inhibitor and the EGFR inhibitor has a great clinical application prospect in EGFR mutant liver cancer cells. A breakthrough point is found for researching a novel method for treating tumors by combining medicaments and improving the clinical medicament treatment effect of the tumors.

At present, the application of the composition in the medicine for treating liver cancer is not reported.

Disclosure of Invention

The invention relates to a new discovery of a drug combination for treating liver cancer, in particular to EGFR (epidermal growth factor receptor) mutant liver cancer, and aims to provide an inhibition effect of a composition of a BET bromodomain inhibitor JQ1 and an EGFR inhibitor Erlotinib on liver cancer tumor cells, so as to achieve an effect of synergistically treating the liver cancer.

The compound a is a BET bromopain inhibitor JQ1, and the structural formula of the compound is (a); the compound b is an EGFR inhibitor Erlotinib, and the structural formula of the compound b is shown as (b). The JQ1 drug is used at a concentration of 50 mg/kg; the Erlotinib drug is used at a concentration of 50 mg/kg. The mass ratio of the compound I to the compound II in the composition is 1:1 to 1:2

Experiments show that compared with the single use of the compound I or the compound II, the composition disclosed by the invention can effectively inhibit the proliferation and the clone formation of EGFR mutant liver cancer cells and inhibit the survival rate of the cells in vivo and in vitro, so that the composition can be used for preparing medicines for treating EGFR mutant tumors.

The BET bromodomain inhibitor JQ1 and the EGFR inhibitor Erlotinib are mixed for use, so that the effects of inhibiting proliferation, clone formation, transfer and the like of liver cancer cells can be effectively achieved. The combined use of the two has obviously improved inhibition effect on the growth of the liver cancer tumor, and can be used for treating EGFR mutant liver cancer

Drawings

FIG. 1JQ1 and EGFR inhibitors are used in combination to inhibit the activity and proliferation of hepatoma cells. (a) Cell viability assay (b) photographing of cell clones and quantitative analysis.

FIG. 2JQ1 and ERL drug action induced apoptosis.

FIG. 3JQ1 in combination with EGFR inhibitors inhibited growth of mouse liver cancer tumors. (a) Tumor volume comparison and tumor volume photographs. (b) And (4) detecting the weight of the mouse tumor after the JQ1 and ERL drugs act.

Detailed Description

The present invention will now be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.

Example 1

Solutions of BET bromodomain inhibitor JQ1 and EGFR inhibitor Erlotinib were prepared.

MYC inhibitor JQ1 and EGFR inhibitor Erlotinib were each dissolved in DMSO to prepare stock solutions with a concentration of 10 mM. JQ1 is diluted to 1 mu M by adding culture solution, and Erlotinib is diluted to 1 mu M by adding culture solution to serve as working concentration;

example 2

After the JQ1 and the EGFR inhibitor are combined to act, the activity and the proliferation of EGFR mutant liver cancer cells are effectively inhibited.

3 x 10 sowing⁴Cells, mixed with Erlotinib with the concentration of 1 μ M diluted by the combined culture solution after being diluted by JQ1 to 1 μ M, 0.1 μ M and 0.01 μ M in the culture solution in a gradient way, cultured EGFR mutant hepatoma cells for 48 hours at 37 ℃, detected by cytotoxicity analysis by the cell Titer Glo method and the clone formation experiment, and then analyzed for significance (T-TEST). The results are shown in fig. 1, the EGFR inhibitor Erlotinib and JQ1 are used in combination, and compared with the JQ1 treatment group with the same concentration, the EGFR inhibitor Erlotinib and JQ1 treatment group can effectively inhibit the activity and proliferation of tumor cells, and the cloning formation number of liver cancer cells is obviously reduced.

Example 3JQ1 and ERL drugs cause apoptosis of EGFR mutant hepatoma cells after action.

3 x 10 sowing⁵Adding 1 mu M JQ1 diluted by a culture solution into the cells, combining the JQ1 diluted by the culture solution with 1 mu M Erlotinib37 degrees to culture and treat the EGFR mutant hepatoma cells for 48 hours, adding 10ul Annexin V-FITC and 5ul PI dyes, incubating for 10min in a dark place, and detecting apoptosis by using a flow cytometer. The results are shown in fig. 2, and the EGFR inhibitor Erlotinib and JQ1 used in combination can effectively cause a large amount of cells to undergo apoptosis compared with the JQ1 and Erlotinib treated groups with the same concentration.

Example 4 combination of JQ1 and EGFR inhibitor is effective in inhibiting growth of mouse liver cancer tumor

MHCC97-H cells were digested, collected and counted, and then resuspended and diluted with PBS to a cell concentration of 5X 10⁷cells/ml PBS. 100ul of matrigel was aspirated and mixed with 100ul of cell sap, and stored on ice. The seeds were implanted subcutaneously into nude mice. After the mice had developed tumors, the mice were divided into 4 groups of 5 eachOnly, the group was divided into a control group, a JQ1(50mg/kg) administration group, an ERL (50mg/kg) administration group and a JQ1(50mg/kg) and ERL (50mg/kg) combined administration group. Administered intraperitoneally once every other day. Nude mice body weights were recorded. The length and width of the tumor were measured every other day with a vernier caliper. Applying a volume formula: v ═ pi/6 ×. length × width 2 mouse tumor volumes were calculated. And (5) drawing a mouse tumor change curve. The results are shown in fig. 3, and the combined use of the two drugs obviously inhibits the growth of the tumor of the mouse compared with the drug without the drug or the drug JQ1 and ERL which are used independently. The size and the weight of the tumor of the combined medicine group are obviously smaller than those of other unused medicine groups and single medicine groups.

The experiments show that the MYC inhibitor JQ1 and the EGFR inhibitor Erlotinib can effectively inhibit the proliferation of tumor cells, the cloning formation of the tumor cells and the survival rate of the tumor cells in cells with EGFR mutation. Therefore, the combined use of the two inhibitors provides a candidate scheme for the development of anti-cancer drugs, and has great clinical application prospect in tumors with EGFR mutation.

Claims

1. An application of a composition of a BET bromodomain inhibitor JQ1 and an EGFR inhibitor Erlotinib in preparing a medicament for treating liver cancer.

2. The use according to claim 1, wherein compound a is the BET bromopain inhibitor JQ1, having the compound formula (a); the compound b is an EGFR inhibitor Erlotinib, and the structural formula of the compound b is shown as (b);

3. the use according to claim 1, wherein the mass ratio of JQ1 to Erlotinib in the composition is 1:1 to 1: 2.

4. The use according to claim 1 or 3, wherein the JQ1 medicament is used at a concentration of 25-50 mg/kg; the using concentration of the Erlotinib medicament is 25-50 mg/kg.

5. The use of claim 1 or 3, wherein JQ1 in combination with Erlotinib is effective in the treatment of EGFR-mutated liver cancer.

6. A medicine for treating liver cancer comprises a BET bromodomain inhibitor JQ1 and an EGFR inhibitor Erlotinib.

7. The drug for treating liver cancer according to claim 6, wherein a pharmaceutically acceptable carrier or adjuvant is further added.