CN106362153A - Application of aldo-keto reductase 1B1 inhibitor in preparation of anti-breast cancer medicines - Google Patents

Abstract

The invention discloses the application of an aldehyde and ketone reductase 1B1 inhibitor in the preparation of anti-breast cancer drugs. The present invention finds that the aldehyde and ketone reductase 1B1 inhibitor can effectively inhibit tumor stem cells, significantly weaken the migration and invasion ability of tumor cells, inhibit the tumorigenesis ability and lung metastasis ability of tumor cells in mice, and can be applied to anti-breast cancer drugs In the preparation of , it can be used for the new indication of targeted therapy of breast cancer.

Description

Application of aldehyde and ketone reductase 1B1 inhibitors in the preparation of anti-breast cancer drugs

technical field

The invention relates to the field of medical technology, in particular to the application of an aldoketone reductase 1B1 inhibitor in the preparation of anti-breast cancer drugs.

Background technique

Breast cancer is a common cancer in women. Although people have studied breast cancer for more than 30 years, about 90% of breast cancer patients still die from local invasion and metastasis of tumor cells, and the average survival time of breast cancer patients after tumor cell metastasis is about 2 years.

The genotypes of breast cancer include luminal subtype A (luminal subtype A), luminal subtype B (luminal subtype B), HER2 overexpression subtype and basal-like subtype. Among all subtypes, basal-like breast cancer has the characteristics of high proliferative activity, easy recurrence, strong invasion and metastasis ability, and has the worst prognosis.

The typical phenotype of basal-like breast cancer is negative for ER, PR and HER2 (triple-negative), and expresses basal cell keratin (CK5/6 and CK14). Basal-like breast cancer may arise from stem cells because of its dual potential for glandular-epithelial and myoepithelial differentiation. Consistent with this observation, basal-like breast cancers have many features of cancer stem cells and markers of epithelial-mesenchymal transition (EMT).

Epithelial-mesenchymal transition (EMT) refers to the transformation of epithelial cells into mesenchymal cells, which plays a key role in embryonic development, tissue remodeling, wound healing and tumor metastasis. Through EMT, cells change from "static" to "moving" and begin to metastasize. This is due to the loss of the intercellular adhesion molecule E-cadherin, the cells detach from the tumor tissue and begin to metastasize and invade surrounding tissues. EMT can endow tumor cells with many characteristics of stem cells, and runs through the entire process of malignant tumor occurrence, development, invasion and metastasis.

Patients with basal-like breast cancer are insensitive to endocrine therapy, easily resistant to chemotherapy, and lack corresponding targeted therapy. Currently, scientists are working hard to find specific basal breast cancer targeting sites, hoping to make a breakthrough in targeted therapy.

Epalrestat (English name is epalrestat), the chemical name is 5-[(1Z,2E)-2-methyl-3-phenyl-2-propenylidene]-4-oxo-2-thio- Thiazolidineacetic acid, molecular formula: C15H13NO3S2, molecular weight: 319.401, structural formula as follows:

Epalrestat is an aldose reductase inhibitor, which is mainly used for the prevention, improvement and treatment of peripheral nerve disorders (numbness, pain) complicated by diabetes, etc. It has also been reported that it can be used for the treatment of diabetic nephropathy. Epalrestat has few side effects and is administered orally. Adults take 50-200 mg each time, three times a day, which is generally considered a safe dose. Adults take 50 mg of this product orally, and the peak blood concentration (3.9 μg/ml) is reached after 1 hour. Animal experiments have confirmed that this product is mainly distributed in the digestive tract, liver and kidneys. After 24 hours, about 8% is excreted through the urethra, and about 80% is excreted from the body by feces.

Contents of the invention

The present invention finds that the aldehyde and ketone reductase 1B1 inhibitor can significantly inhibit the occurrence, invasion and metastasis of breast cancer, and can be used for preparing anti-breast cancer drugs.

In the present invention, 51 breast cancer cell lines of different subtypes were analyzed by gene chips, and we found that the expression level of aldoketone reductase 1B1 (AKR1B1) in basal-like breast cancer cells was significantly higher than that of other subtypes of cells.

Furthermore, through TCGA data analysis, it was found that the expression level of AKR1B1 in triple-negative breast cancer was also significantly higher than that in other subtypes. Studies have found that AKR1B1 helps to promote the occurrence and development of breast cancer; and AKR1B1 inhibitors can significantly inhibit the occurrence, invasion and metastasis of breast cancer.

The aforementioned triple-negative breast cancer refers to breast cancer that is negative for ER, PR, and HER2 receptors, and is highly consistent with basal-like breast cancer.

The invention provides the application of epalrestat in the preparation of anti-basal-like breast cancer drugs.

The present invention takes epalrestat, an inhibitor of aldehyde and ketone reductase 1B1, as an example to carry out experiments, using breast cancer cell lines (SUM159 and MDA-MB231) as cell models, and using tumor stem cell detection technology to detect the inhibition of epalrestat on tumor stem cells The effect of epalrestat on cell tumorigenesis was detected in vitro by soft agar colony formation method, and the inhibitory effect of epalrestat on cell migration and invasion was detected in vitro by cell migration and cell invasion method; the results found that epalrestat It has an inhibitory effect on tumor stem cells, and has effects on cell tumorigenesis, cell migration and invasion.

In addition, the present invention uses a mouse model of tumor formation and tumor lung metastasis to detect the inhibitory effect of epalrestat on tumor formation and metastasis in vivo, which also proves that epalrestat can significantly inhibit the tumorigenesis ability of tumor cells in mice and the lung transfer ability.

The invention provides an anti-breast cancer drug, which comprises an aldoketone reductase 1B1 inhibitor.

The present invention also provides an anti-basal-like breast cancer drug, which comprises an aldoketone reductase 1B1 inhibitor.

The invention also provides an anti-triple-negative breast cancer drug, which comprises an aldoketone reductase 1B1 inhibitor.

Preferably, the aldehyde and ketone reductase 1B1 inhibitor is epalrestat.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention finds that the aldehyde and ketone reductase 1B1 inhibitor can effectively inhibit tumor stem cells, significantly weaken the migration and invasion ability of tumor cells, inhibit the tumorigenesis ability and lung metastasis ability of tumor cells in mice, and can be applied to anti-breast cancer drugs In the preparation of , it can be used for the new indication of targeted therapy of breast cancer.

Description of drawings

Figure 1 is a graph showing the expression of AKR1B1 in 51 breast cancer cell lines of different subtypes.

Figure 2 is an effect diagram of the expression of AKR1B1 in 349 breast cancer samples of different subtypes.

Fig. 3 is an effect diagram of epalrestat inhibiting the sphere formation ability of breast cancer cells.

Fig. 4 is a diagram showing the effect of epalrestat inhibiting the ability of soft agar colony formation of breast cancer cells.

Fig. 5 is an effect diagram of epalrestat inhibiting the migration ability of breast cancer cells.

Fig. 6 is an effect diagram of epalrestat inhibiting the invasion ability of breast cancer cells.

Fig. 7 is a diagram showing the effect of epalrestat on inhibiting the tumorigenic ability of tumor cells in mice.

Fig. 8 is a graph showing the effect of epalrestat on inhibiting the ability of tumor cells to metastasize to the lungs of mice.

detailed description

The present invention will be further described below in combination with specific implementation cases.

The epalrestat involved in the following examples were all purchased from Shandong Dyne Marine Biopharmaceutical Co., Ltd. (epalrestat tablets sold on the market can also be used); the involved "SUM159 and MDA-MB231" two breast cancer strains The cell lines were donated by Peter Zhou's laboratory at the University of Kentucky, and can also be purchased on the market. The above cell lines are only used for experimental verification and do not involve cell preservation.

Example 1 Expression of AKR1B1 in different subtypes of breast cancer cell lines

In order to detect the expression of aldoketone reductase 1B1 (AKR1B1) in different subtypes of breast cancer cell lines, the gene expression of 51 breast cancer cell lines cDNA library (GSE12777) was analyzed. The differences in AKR1B1 expression between 23 basal breast cancer cell lines and 28 other subtype cell lines were compared by T test.

Results As shown in Figure 1, the expression level of AKR1B1 in basal-like breast cancer cells was significantly higher than that in other subtypes of cells.

Example 2 Expression of AKR1B1 in different subtypes of breast cancer samples

In order to detect the expression of AKR1B1 in different subtypes of breast cancer, the gene expression of 349 breast cancer tissue samples cDNA library (TCGA) was analyzed. The differences in AKR1B1 expression between 49 triple-negative breast cancer samples and 300 other subtype samples were compared by T test.

The results are shown in Figure 2, the expression level of AKR1B1 in triple-negative breast cancer was significantly higher than that in other subtypes.

(Note: Breast cancer cell line cDNA library (GSE12777) and breast cancer tissue sample cDNA library (TCGA) can be accessed through http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12777 and https://tcga-data.nci.nih.gov/tcga/dataAccessMatrix.htm.)

Example 3 Inhibitory effect of epalrestat on breast cancer stem cells

(1) Take breast cancer cells in the logarithmic growth phase and blow them into single cells, use stem cell medium to make a concentration of 20,000 cells/mL, take 2 mL and add them to a non-adherent 6-well plate for tumor stem cell sphere formation (mammoshere )experiment;

(2) Two breast cancer cell lines, SUM159 and MDA-MB231, were selected as cell models;

(3) Dosage: the final concentration is 20mM epalrestat, and the control group is the same volume of solvent DMSO; time of administration: when the cells are added to a non-adherent 6-well plate, the cycle is 10 days; Optical microscopes were used to observe and count, and to analyze statistics.

Results As shown in Figure 3, epalrestat significantly inhibited the sphere-forming ability of tumor cells.

Example 4 Inhibitory effect of epalrestat on tumorigenesis of breast cancer cells in vitro

(1) The breast cancer cells in the logarithmic growth phase were blown into single cells, and the concentration was 10,000 cells/mL, and they were inoculated in 24-well culture dishes for Soft-Agar Colony-formation Assay (Soft-Agar Colony-formation Assay). );

(2) Two breast cancer cell lines, SUM159 and MDA-MB231, were selected as cell models;

(3) Dosage: the final concentration is 20mM epalrestat, and the control group is the same volume of solvent DMSO; administration time: once every 3 days when the liquid is changed, and the cycle is 2 weeks;

(4) Invert the plate and superimpose a transparent film with a grid, directly count the clones with the naked eye, or count the clones larger than 10 cells under a microscope (low magnification lens); finally calculate the clone formation rate.

Results As shown in Figure 4, epalrestat significantly inhibited the ability of tumor clone formation.

Example 5 Inhibitory effect of epalrestat on breast cancer cell migration in vitro

(1) Take the breast cancer cells in the logarithmic growth phase and blow them into single cells, make a concentration of 500,000 cells/mL, and inoculate 50ul/well in the chemotaxis chamber for cell migration experiments;

(2) Two breast cancer cell lines, SUM159 and MDA-MB231, were selected as cell models;

(3) Administration dose: the final concentration is 20mM epalrestat, and the control group is the solvent DMSO of the same volume; administration time: administration before the preparation of cells;

(4) Crystal violet staining, counting under a microscope (low magnification).

Results As shown in Figure 5, epalrestat significantly inhibited the migration ability of tumor cells.

Example 6 Epalrestat inhibits breast cancer cell invasion in vitro

(1) Take the breast cancer cells in the logarithmic growth phase and pipette them into single cells, make a concentration of 500,000 cells/mL, and inoculate 50ul/well in a tranwell chamber for cell invasion experiments;

(2) Two breast cancer cell lines, SUM159 and MDA-MB231, were selected as cell models;

(3) Administration dose: the final concentration is 20mM epalrestat, and the control group is the solvent DMSO of the same volume; administration time: administration before the preparation of cells;

(4) Crystal violet staining, counting under a microscope (low magnification).

Results As shown in Figure 6, epalrestat significantly inhibited the invasion ability of tumor cells.

Example 7 Inhibitory Effect of Epalrestat on the Tumor Formation Ability of Tumor Cells in Mice

(1) 6-week-old female NOD/SCID mice were selected, and 1×10 ⁶ MDA-MB231 cells were injected into the left mammary fat pad of the mice, and the growth of tumor cells was observed.

(2) Dosage: administered by intragastric administration, the dose of epalrestat was 50 mg/kg/day, and the control group was the same volume of solvent water; administration time: administered every morning, a total of 4-5 weeks;

(3) The size of the tumor was measured every 2 days, and a growth curve was drawn; after the mice were sacrificed, the tumors of the experimental group and the control group were weighed.

Results As shown in Figure 7, epalrestat significantly inhibited the tumorigenic ability of tumor cells in mice.

Example 8 Inhibitory Effect of Epalrestat on the Lung Metastasis Ability of Tumor Cells in Mice

(1) 6-week-old female NOD/SCID mice were selected, and 1×10 ⁶ MDA-MB231 cells stably expressing luciferase were injected into the tail vein of the mice;

(2) Dosage: administered by intragastric administration, the dose of epalrestat was 50 mg/kg/day, and the same volume of solvent water was used in the control group; administration time: administered every morning for a total of 4 weeks;

(3) Four weeks later, the mouse lung metastasis was detected with IVIS-100 imaging system.

The results are shown in Figure 8, epalrestat treatment significantly inhibited the ability of tumor cells to metastasize to the lungs of mice.

Claims (9)

1. application in preparing anti-breast cancer medicines for the aldehyde ketone reductase 1b1 inhibitor.

2. application in preparing anti-breast cancer medicines for the epalrestat.

3. application in preparing anti-substrate sample breast cancer medicines for the epalrestat.

4. a kind of anti-breast cancer medicines are it is characterised in that comprise aldehyde ketone reductase 1b1 inhibitor.

5. anti-breast cancer medicines as claimed in claim 4 it is characterised in that described aldehyde ketone reductase 1b1 inhibitor be according to Pa Sita.

6. a kind of anti-substrate sample breast cancer medicines are it is characterised in that comprise aldehyde ketone reductase 1b1 inhibitor.

7. substrate sample breast cancer medicines as claimed in claim 6 are it is characterised in that described aldehyde ketone reductase 1b1 inhibitor For epalrestat.

8. a kind of anti-triple negative breast cancer medicine is it is characterised in that comprise aldehyde ketone reductase 1b1 inhibitor.

9. triple negative breast cancer medicine as claimed in claim 8 is it is characterised in that described aldehyde ketone reductase 1b1 inhibitor For epalrestat.