Application of sodium glycochenodeoxycholate in the preparation of drugs for treating breast cancer

Abstract

The present invention provides the use of sodium glycochenodeoxycholate in the preparation of a medicament for treating breast cancer, belonging to the technical field of breast cancer treatment. The present invention first proposes that sodium glycochenodeoxycholate has significant biological activity in inhibiting the proliferation, metastasis and invasion of breast cancer cells, can significantly inhibit the growth, spread and invasion of breast cancer cells, thereby improving the treatment effect of breast cancer, and can reduce the risk of progression and metastasis of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of breast cancer treatment, and particularly relates to the application of glycochenodeoxycholic acid sodium in the preparation of drugs for treating breast cancer. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women, and liver metastasis of breast cancer has a high incidence and mortality. The occurrence and development of breast cancer are related to cell proliferation, metastasis and invasion. The metastasis of breast cancer cells remains a difficult problem in the treatment of breast cancer. Therefore, searching for drugs with inhibitory effects on the proliferation and metastasis of breast cancer cells has important clinical application value. Glycochenodeoxycholic acid sodium (also known as glycochenodeoxycholate) is a compound, and there is no relevant report in the prior art on whether it has the effect of treating breast cancer. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide the application of glycochenodeoxycholic acid sodium in the preparation of drugs for treating breast cancer, and it is first proposed that glycochenodeoxycholic acid sodium has the effects of efficiently inhibiting the proliferation, metastasis and invasion of breast cancer cells, and can improve the treatment effect of breast cancer.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides the application of glycochenodeoxycholic acid sodium in the preparation of drugs for treating breast cancer.

[0006] The present invention also provides the application of glycochenodeoxycholic acid sodium in the preparation of drugs for inhibiting the proliferation of breast cancer cells.

[0007] The present invention also provides the application of glycochenodeoxycholic acid sodium in the preparation of drugs for inhibiting the migration of breast cancer cells.

[0008] The present invention also provides the application of glycochenodeoxycholic acid sodium in the preparation of drugs for preventing and treating the invasion of breast cancer cells.

[0009] The present invention also provides a drug for treating breast cancer, and the drug comprises glycochenodeoxycholic acid sodium and pharmaceutically acceptable excipients.

[0010] Preferably, the drug contains 1-90% of the active ingredient glycochenodeoxycholic acid sodium.

[0011] Preferably, the dosage form of the drug includes injection, tablet, capsule or granule.

[0012] Preferably, the injection includes intravenous injection and subcutaneous injection.

[0013] Advantages of the present invention:

[0014] The present invention first proposes that sodium glycochenodeoxycholate has significant biological activity in inhibiting the proliferation, metastasis and invasion of breast cancer cells, can significantly inhibit the growth, spread and invasion of breast cancer cells, thereby improving the treatment effect of breast cancer, and can also reduce the risk of progression and metastasis of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the structural formula of sodium glycochenodeoxycholate of the present invention;

[0016] Figure 2 is the result of CCK-8 cell proliferation assay;

[0017] Figure 3 is the result of scratch assay of MDA-MB-231 cells;

[0018] Figure 4 is the result of scratch assay of MCF-7 cells;

[0019] Figure 5 is the result of Transwell invasion and migration assays of two breast cancer cell lines;

[0020] Figure 6 is the statistical result of Transwell invasion assay data of two breast cancer cell lines;

[0021] Figure 7 is the result of tumorigenesis in nude mice intervened with sodium glycochenodeoxycholate in MDA-MB-231, where A, B, and C are control pictures of tumorigenesis in nude mice, D is the growth chart of tumor volume, and E is the statistical chart of tumor weights of two groups of nude mice;

[0022] Figure 8 is the result of HE staining and Ki67 detection of tumorigenesis in nude mice of MDA-MB-231. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides the use of sodium glycochenodeoxycholate in the preparation of drugs for treating breast cancer.

[0024] The present invention also provides the use of sodium glycochenodeoxycholate in the preparation of drugs for inhibiting the proliferation, migration and / or invasion of breast cancer cells.

[0025] The present invention has no special limitation on the specific source of sodium glycochenodeoxycholate, which can be synthesized by conventional chemical synthesis methods in the art. Sodium glycochenodeoxycholate has good preparability and stability, and can also be obtained by purchasing through commercial channels. The structural formula of the glycochenodeoxycholic acid described in the present invention is as Figure 1As shown. In the present invention, the breast cancer cells preferably include MDA-MB-231 cells and MCF-7 cells. In the present invention, when preparing a drug for treating breast cancer with chenodeoxycholic acid sodium glycine, it can be used as the sole active ingredient of the drug, or can be combined with other active ingredients having the effect of treating breast cancer to prepare a drug.

[0026] The present invention also provides a drug for treating breast cancer, and the drug includes chenodeoxycholic acid sodium glycine and pharmaceutically acceptable excipients.

[0027] The present invention has no special limitation on the types of pharmaceutically acceptable excipients, and conventional pharmaceutically acceptable excipients in the art can be used. In the present invention, the drug preferably contains 1-90% of the active ingredient chenodeoxycholic acid sodium glycine, and more preferably contains 10-80% of the active ingredient chenodeoxycholic acid sodium glycine, where the percentage represents the weight ratio of the active ingredient to the drug. In the present invention, the drug can be used for single-drug treatment, or can be used in combination with other drugs having the effect of treating breast cancer for treatment. In the present invention, the dosage form of the drug preferably includes injection, tablet, capsule or granule, and the injection preferably includes intravenous injection and subcutaneous injection. The drug of the present invention can be applied to clinical practice through various administration routes such as oral administration, intravenous injection, subcutaneous injection, etc., which has great flexibility and convenience, and using chenodeoxycholic acid sodium glycine as the drug active ingredient can produce low drug resistance to breast cancer cells, thereby reducing the occurrence of treatment failure and recurrence.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they cannot be understood as limiting the protection scope of the present invention.

[0029] In the following embodiments, unless otherwise specified, all are conventional methods.

[0030] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0031] Example 1

[0032] IC50 experiment

[0033] Take breast cancer cells MDA-MB-231 and MCF-7 in the logarithmic growth phase, digest them with trypsin and then centrifuge, count the cell density to be about 1×10 5 cells / ml, and evenly spread about 100 μl of the cell suspension in each well of a 96-well plate, with 5 replicate wells for each sample.

[0034] The 96-well plates were placed in an incubator at 37 °C with 5% CO2. After culturing for 24 h until the cells adhered to the plate, glycochenodeoxycholic acid sodium salt at different concentrations (100, 200, 300, 400, 500, 600, 700, 800, 900 μg / ml) (purchased from SIGMA, product number G0759-100MG) was added. After culturing the cells for 24 and 48 hours, 10 μl of CCK8 solution was added to each well, and the 96-well plates were gently tapped to assist in mixing evenly. Then they were placed in an incubator at 37 °C with 5% CO2 and incubated for 1 - 4 h. The absorbance at 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader at each time point (1 h, 2 h, 3 h, 4 h), and the IC50 concentration was calculated. The results showed that the IC50 of glycochenodeoxycholic acid sodium salt for MDA-MB-231 cells at 4 h was 298.232 μg / ml, and the IC50 of glycochenodeoxycholic acid sodium salt for MCF-7 cells at 4 h was 442.77.

[0035] Example 2

[0036] CCK-8 cell proliferation assay

[0037] Breast cancer cells MDA-MB-231 and MCF-7 in the logarithmic growth phase were taken, digested with trypsin and then centrifuged. The cell density was counted to be about 1×10 5 / ml. The cell suspension was evenly spread in 96-well plates at about 100 μl per well, with 4 replicate wells for each sample. There were blank groups (DMEM medium), control groups (untreated cells), and experimental groups (glycochenodeoxycholic acid sodium salt added: 200 μg / ml was added to MDA-MB-231 cells, and 300 μg / ml was added to MCF-7 cells).

[0038] The 96-well plates were placed in an incubator at 37 °C with 5% CO2. After culturing for 24 h until the cells adhered to the plate, glycochenodeoxycholic acid sodium salt compounds were added. After culturing the cells for 24 hours, it was set as the 0 point, and the proliferation ability of the cells at 0 h, 24 h, 48 h, 72 h, etc. was detected.

[0039] At each detection point, 10 μl of CCK8 solution was added to each well, and the 96-well plates were gently tapped to assist in mixing evenly. Then they were placed in an incubator at 37 °C with 5% CO2 and incubated for 4 h. The absorbance at 450 nm was detected using an ELISA reader, and data calculations were performed. The results were as Figure 2 shown, and glycochenodeoxycholic acid sodium salt had a significant effect on inhibiting the proliferation of breast cancer cells.

[0040] Example 3

[0041] Cell scratch assay to detect the effect of glycochenodeoxycholic acid sodium salt compounds on the migration ability of breast cancer cells

[0042] Breast cancer cells MDA-MB-231 and MCF-7 that had grown to about 80%. After digestion with trypsin and centrifugation, the cell density was counted to be 3.5×105 Around / ml, the number of cells should be such that they can cover the surface after overnight adherence. Spread the cell suspension evenly in a 6-well plate at about 2 ml per well, with 3 replicate wells for each sample.

[0043] Use a marker pen to draw a vertical line every 1 cm on the back of the 6-well plate, crossing the wells horizontally, for easy microscopic observation. After the cells have grown, the next day, use a 200-μl sterile pipette tip to make a "one"-shaped scratch on the culture plate, perpendicular to the horizontal lines on the back, and try to ensure that each scratch has the same width. Gently rinse with PBS.

[0044] Add serum-free medium to the 6-well plate. The control group is untreated cells (NC), and the experimental groups (Treat, T) are added with the corresponding concentration of glycochenodeoxycholic acid (the added concentration for MDA-MB-231 cells is 200 μg / ml, and the added concentration for MCF-7 cells is 300 μg / ml). Place the scratched 6-well plate in a cell culture incubator at 37 °C and 5% CO2 for culture. Take images at 0 h, 12 h, 24 h, and 36 h after scratching using an Olympus inverted phase contrast microscope at 100× magnification. Some of the results are as Figure 3 and Figure 4 shown. Compared with the control group, the scratch migration of MDA-MB-231 cells in the experimental group slowed down after 24 h and 36 h, with statistically significant differences. The scratch migration of MCF-7 cells in the experimental group slowed down after 24 h, with statistically significant differences.

[0045] Example 4

[0046] Transwell (Corning pore size: 8 μm) invasion and migration assay

[0047] Dilute Matrigel at a ratio of 1:8. Coat the upper surface of the bottom membrane of the Transwell chamber with the diluted solution and place it at 37 °C for 30 min to allow Matrigel to form a gel. Hydrate the basement membrane before use for invasion assays. Do not coat with Matrigel for migration assays.

[0048] Prepare cell suspension

[0049] First, starve the cells of serum for 24 h to remove the influence of serum. The experimental group and the control group (the control group (NC) is the group of cells untreated with drugs, and the experimental group (T) is the group of cells treated with glycochenodeoxycholic acid, with the added concentration for MDA-MB-231 cells being 200 μg / ml and the added concentration for MCF-7 cells being 300 μg / ml)) have the same cell state, both under normal culture conditions with a cell confluence of 80%. Digest the cells into single cells, centrifuge and discard the culture medium after terminating digestion, wash once with PBS, perform cell counting, and resuspend with serum-free medium without FBS. Adjust the cells to 2.5×105 cells / ml.

[0050] Inoculated cells

[0051] Take 2.5×10 5 μl of cell suspension was added to the upper chamber of a Transwell insert in serum-free DMEM. Approximately 800 μl of medium containing 20% FBS was added to the lower chamber of a 24-well plate. Bubbles were likely to form between the lower culture medium and the insert, and bubble formation was avoided as much as possible. The 24-well plate was placed in a 37 °C, 5% CO₂ cell culture incubator and cultured routinely for 24 h.

[0052] Result statistics

[0053] The Transwell insert was taken out, the medium in the wells was aspirated completely, washed twice with PBS, fixed with methanol for 20 min, and the insert was air-dried. The membrane was removed, stained with 0.5% crystal violet solution for 20 min, the upper-layer cells were gently wiped off with a cotton swab, and washed three times with PBS. Observation and photography were performed under a microscope. The results were as Figure 5 and Figure 6 shown. The number of cells passing through the Transwell insert in the experimental group was significantly lower than that in the control group, indicating that glycochenodeoxycholic acid could inhibit the invasion of breast cancer cells.

[0054] Example 5

[0055] Animal experiment

[0056] In this experiment, immunodeficient female nude mice (BALB / c), 5 weeks old (about 17±1 g / each), of SPF grade, were provided by the Department of Medical Experimental Animal Center of Guangxi Medical University. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council). All experimental protocols and operating procedures complied with the regulations of the Animal Use and Management Committee of the Medical Experimental Animal Center of Guangxi Medical University.

[0057] MDA-MB-231 cells to be used in animal experiments were seeded in a T75 culture flask after the same cell counting. When the cells grew to 80%, the culture medium was discarded, the cells were washed twice with 3 ml of PBS, digested with 0.5% trypsin for 3 min, and the digestion was terminated with 10% FBS culture medium. The cells were then transferred to a 15-ml centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, 3 ml of PBS was added, and the cell concentrations of the experimental group and the control group were adjusted to 7.95×10 6 cells / mL.

[0058] 200 μl of cell suspension (with air expelled) was aspirated with a 200-μl insulin syringe and injected into the right axilla of nude mice; 5 nude mice were in each group;

[0059] Observe the body weight and status of nude mice once a day. After the nude mice developed tumors (subcutaneous tumor implantation was given 5 days after adaptation in the animal room since the purchase date, and tumors were found on the 10th day), inject sodium glycochenodeoxycholate intraperitoneally into the nude mice in the experimental group once every 3 days (intraperitoneal injection was given on the 14th, 17th, and 20th days), and the dosage was 5 mg / kg. Inject sterilized PBS solution intraperitoneally into the nude mice in the control group once every 3 days (intraperitoneal injection was given on the 14th, 17th, and 20th days), and the dosage was 5 mg / kg. Observe the body weight and status of nude mice and measure the tumor volume once every 3 days. The calculation formula is tumor volume (mm 3 ) = length × width 2 × 0.5. On the 23rd day, decapitate the nude mice and remove the transplanted tumors, and measure the tumor weight. The results are as Figure 7 shown. It can be Figure 7 seen that sodium glycochenodeoxycholate can inhibit the proliferation of murine tumors in the nude mouse model.

[0060] Cut the tumor along the largest cross-section and immerse it in neutral formalin solution with 20 times the volume. After fixation for 24 hours, dehydrate the tissue programmatically and embed it in paraffin blocks for standby. Perform HE staining on it, and detect the positive rate of Ki67 (proliferation index) from the aspect of immunohistochemistry to further verify the effect of sodium glycochenodeoxycholate in inhibiting the proliferation of murine tumors in the nude mouse model. The specific method is as follows:

[0061] HE staining

[0062] Deparaffinize the paraffin sections to water: sequentially place the sections in xylene Ⅰ for 20 min - xylene Ⅱ for 20 min - absolute ethanol Ⅰ for 5 min - absolute ethanol Ⅱ for 5 min - 75% alcohol for 5 min, and wash with tap water.

[0063] Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3 - 5 min, wash with tap water, differentiate with differentiating solution, wash with tap water, blue with bluing solution, and rinse with running water.

[0064] Eosin staining: Immerse the sections in gradient alcohols of 85% and 95% for dehydration for 5 min each, and stain in eosin staining solution for 5 min.

[0065] Dehydration and mounting: Sequentially place the sections in absolute ethanol I for 5 min - absolute ethanol II for 5 min - absolute ethanol III for 5 min - xylene Ⅰ for 5 min - xylene Ⅱ for 5 min for transparency, and mount with neutral gum.

[0066] Microscopic examination and image acquisition and analysis.

[0067] Immunohistochemical staining

[0068] (1) Deparaffinization and hydration of sections: Immerse in xylene I for 20 minutes; immerse in xylene II for 10 minutes; immerse in xylene III for 10 minutes; immerse in 100% alcohol I for 10 minutes; immerse in 100% alcohol II for 10 minutes; immerse in 100% alcohol III for 10 minutes; immerse in 90% alcohol for 10 minutes; immerse in 80% alcohol for 10 minutes; immerse in 70% alcohol for 10 minutes. Rinse with PBS 2 times, 1 minute each time.

[0069] (2) Antigen retrieval: Place the sections in a microwave oven with EDTA (pH 9.0), microwave on medium heat for 8 minutes, stop for 8 minutes, then turn to medium-low heat for 7 minutes, and let it cool naturally. Then place the slides in PBS (pH 7.4) and shake on a decolorizing shaker for 3 washes, 5 minutes each time.

[0070] (3) Block endogenous peroxidase: Put the sections into 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Then place the slides in PBS (pH 7.4) and shake on a decolorizing shaker for 3 washes, 5 minutes each time. Rinse with PBS 3 times, 3 minutes each time.

[0071] (4) Dropwise add serum blocking solution within the immunohistochemical circle, evenly cover the tissue, and block at room temperature for 30 minutes. Discard the blocking solution without washing.

[0072] (5) Dropwise add Ki67 primary antibody (concentration 1:1000) and incubate overnight in a wet box at 4°C.

[0073] (6) Take out the next day and let it stand at room temperature for 30 minutes. Place the slides in PBS (pH 7.4) and shake on a decolorizing shaker for 3 washes, 5 minutes each time. After gently blotting dry the sections, dropwise add goat anti-rabbit IgG HRP-labeled secondary antibody corresponding to the primary antibody within the circle to cover the tissue, and incubate at room temperature for 50 minutes. Rinse with PBS 3 times, 3 minutes each time.

[0074] (7) DAB color development: After gently blotting dry the sections, dropwise add freshly prepared DAB color development solution within the circle, control the color development time under the microscope, positive is brownish-yellow, and rinse the sections with tap water to terminate the color development.

[0075] (8) Counterstain the cell nuclei: Counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiating solution for a few seconds, rinse with tap water, blue with hematoxylin bluing solution, and rinse with running water.

[0076] (9) Dehydration and mounting: Sequentially immerse the sections in 75% alcohol for 5 minutes - 85% alcohol for 5 minutes - absolute ethanol I for 5 minutes - absolute ethanol II for 5 minutes - n-butanol for 5 minutes - xylene I for 5 minutes for dehydration and clearing. Take out the sections from xylene, let it dry slightly, and mount with neutral resin.

[0077] (10) Image acquisition and optical density analysis: The cell nuclei were stained blue with hematoxylin, and the positive expression shown by DAB was brownish yellow. Images were taken using a microscope and the integrated optical density values were analyzed using Image Pro Plus software to determine the expression level of Ki67 protein.

[0078] The results were as Figure 8 shown, and chenodeoxycholic acid sodium could inhibit the proliferation of murine tumors in the nude mouse model.

[0079] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of sodium glycochenodeoxycholate in the preparation of a drug for inhibiting the proliferation of breast cancer cells, characterized in that, The breast cancer cells are MDA-MB-231 cells.

2. Use of sodium glycochenodeoxycholate in the preparation of a drug for inhibiting the migration of breast cancer cells, characterized in that, The breast cancer cells are MDA-MB-231 cells.

3. Use of sodium glycochenodeoxycholate in the preparation of a drug for preventing and treating breast cancer cell invasion, characterized in that, The breast cancer cells are MDA-MB-231 cells.

4. The application according to any one of claims 1 to 3, characterized in that, The drug includes sodium glycochenodeoxycholate and pharmaceutically acceptable excipients; the drug contains 1-90% of the active ingredient sodium glycochenodeoxycholate.

5. The application according to claim 4, wherein The dosage form of the drug includes injection, tablet, capsule or granule.

6. The application according to claim 5, wherein The injection includes intravenous injection and subcutaneous injection.

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