Breast cancer cell inhibitor containing 8-O-acetyl shanzhiside methyl ester and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a breast cancer cell inhibitor containing 8-O-acetyl shanzhiside methyl ester and application of the breast cancer cell inhibitor. The concentration of the 8-O-acetyl shanzhiside methyl ester in the inhibitor is 100 [mu] M to 200 [mu] M. The active component 8-O-acetyl shanzhiside methyl ester in the inhibitor not only has a good inhibition effect on proliferation and migration of triple-negative breast cancer cells, but also can effectively inhibit invasiveness and clone formation ability of the triple-negative breast cancer cells, and can be used as a low-toxicity and high-efficiency natural treatment candidate for subtype of triple-negative breast cancer lacking targeted drugs. The chemotherapy sensitivity is enhanced, and the significance in related research and clinical application of breast cancer is greater.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a breast cancer cell inhibitor containing 8-O-acetylganoside methyl ester and its application. Background Technology

[0002] Breast cancer is a malignant tumor originating from the epithelial tissue of the breast. It is the most common malignant tumor among women worldwide, accounting for approximately 23.8% of all new female cancer cases, seriously threatening women's health.

[0003] Based on molecular subtyping, breast cancer can be mainly classified into hormone receptor-positive, HER2-positive, and triple-negative breast cancer. Triple-negative breast cancer (TNBC) accounts for approximately 15%-20% of cases, characterized by negativity of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2). Therefore, it lacks a clear therapeutic target, and standard endocrine therapy (such as tamoxifen) and HER2-targeted therapy (such as trastuzumab) are completely ineffective. Furthermore, TNBC typically exhibits higher histological grade, a higher cell proliferation index, and greater invasiveness. Compared to other subtypes, TNBC is more prone to early metastasis, especially to the lungs and central nervous system; clinical statistics show that approximately 15%-30% of patients develop brain metastases, and its recurrence rate is also significantly increased. Therefore, TNBC has limited treatment options, a very poor prognosis, and a typically low 5-year survival rate, posing a major challenge in diagnosis and treatment.

[0004] Traditional Chinese medicine (TCM), as an important component of comprehensive cancer treatment, demonstrates unique advantages in improving patients' clinical symptoms due to its holistic regulation and multi-target effects. Studies have found that various TCM components exhibit inhibitory effects on tumors, such as paclitaxel, berberine, triptolide, and curcumin. The use of TCM components in the treatment of triple-negative breast cancer is also an important area of ​​current cancer research, playing a role in reducing toxicity and enhancing efficacy, regulating immunity, and inhibiting metastasis.

[0005] 8-O-Acetyl shanzhiside methylester (Barlerin) is a naturally occurring iridoid glycoside compound. It is mainly extracted and isolated from the leaves of the Tibetan medicinal plant *Lamiophlomisrotata Kudo*, and is the main active ingredient of *Lamiophlomisrotata Kudo*.

[0006] Barlerin possesses broad biological activity, exerting protective effects on neurodegenerative disease models through multiple pathways, including anti-inflammatory and antioxidant effects. It effectively inhibits the expression of inflammatory factors, showing improvement in both acute and chronic inflammation models, and exhibiting certain central and peripheral analgesic effects. However, its antitumor mechanism and in vivo efficacy still require further investigation.

[0007] Currently, there are no research reports on the effects of Barlerin on breast cancer (especially triple-negative breast cancer). Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a breast cancer cell inhibitor containing 8-O-acetylganoside methyl ester and its application.

[0009] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The present invention provides a breast cancer cell inhibitor containing 8-O-acetylganoside methyl ester, wherein the concentration of 8-O-acetylganoside methyl ester is 100μM-200μM.

[0010] Based on the above technical solution, the present invention can be further improved as follows.

[0011] Furthermore, the solvent for the inhibitor is a cell culture medium containing 0.1% dimethyl sulfoxide.

[0012] The present invention also provides the use of the breast cancer cell inhibitor containing 8-O-acetylganoside methyl ester as described above in the preparation of a medicament for treating breast cancer.

[0013] Furthermore, the breast cancer cells are triple-negative breast cancer cells.

[0014] Furthermore, the triple-negative breast cancer cells are MDA-MB-231 cells.

[0015] The present invention also provides a medicament for preparing a treatment for breast cancer, the medicament comprising the breast cancer cell inhibitors described above.

[0016] Furthermore, the drug also includes other pharmaceutically acceptable excipients, including at least one of diluents, binders, absorbents, disintegrants, dispersants, humectants, solubilizers, buffers, and surfactants.

[0017] Furthermore, the drug is one or more of the formulations, namely tablets, powders, and liquids.

[0018] The beneficial effects of this invention are as follows: The breast cancer cell inhibitor of the present invention contains 8-O-acetylganoside methyl ester (Barlerin), the active ingredient of which can not only effectively inhibit the cell viability, proliferation and migration of breast cancer cells, but also effectively inhibit their invasiveness and colony formation. It also inhibits tumors by inhibiting the EGFR (epidermal growth factor receptor) related signaling pathway, thereby inhibiting the expression of oncogenic proteins HIF-1α and c-Myc, thus achieving comprehensive and effective inhibition of breast cancer cells. Attached Figure Description

[0019] Figure 1 This is a graph showing the cell viability test results after treatment with various concentrations of Barlerin in Example 1 of the present invention, wherein, ** P< 0.01, **** P< 0.001; Figure 2 The results of the cell proliferation experiment in Example 2 of the present invention; Figure 2 In the middle A, there are EdU staining images after treatment with various concentrations of Barlerin; Figure 2 Figure B shows the statistical results of the proportion of EdU-positive cells; among them, ** P< 0.01, *** P< 0.001, **** P< 0.0001; Figure 3 The results of the cell migration ability test in Example 3 of the present invention; Figure 3 Image A shows crystal violet staining of migrating cells after treatment with various concentrations of Barlerin; Figure 3 Image B shows the cell count results of migrated MDA-MB-231 cells; among them, *** P< 0.001, **** P< 0.0001; Figure 4 The results of the invasion experiment in Example 4 of the present invention; Figure 4 Image A shows crystal violet staining of invasive cells after treatment with various concentrations of Barlerin; Figure 4 Image B shows the cell count results of MDA-MB-231 cells after invasion; where, ** P< 0.01, *** P< 0.001, **** P< 0.0001; Figure 5 The results of the clone formation experiment in Example 5 of the present invention; Figure 5 In Figure A, the results of crystal violet staining of cell colonies after treatment with various concentrations of Barlerin are shown. Figure 5 Image B shows the colony count results of MDA-MB-231 cells; where, ** P<0.01, *** P< 0.001; Figure 6 The results of network pharmacology analysis in Example 6 of the present invention are as follows. Figure 6 In the middle, A is the Venn diagram generated by the intersection of Barlerin target and breast cancer target; Figure 6 Figure B shows the results of PPI network construction and core target selection; Figure 6 The graph in C represents the results of the GO functional enrichment analysis; Figure 6 Figure D in the middle is the result of KEGG functional enrichment analysis; Figure 7 The test results are from Example 7 of the present invention. Figure 7 Figure A shows the expression of epidermal growth factor receptor EGFR and oncogenic proteins HIF-1α and c-Myc in MDA-MB-231 cells after treatment with different concentrations of Barlerin. Figure 7 B in the figure represents the statistical analysis chart of the gray values ​​of the EGFR protein band; Figure 7 The image in C represents a statistical analysis of the grayscale values ​​of the HIF-1α protein band. Figure 7 The image in the middle (D) represents the statistical analysis of the grayscale values ​​of the c-Myc protein band; where * P< 0.05, ** P< 0.01, **** P< 0.0001. Detailed Implementation

[0020] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0021] The present invention relates to a breast cancer cell inhibitor containing 8-O-acetylganoside methyl ester, wherein the concentration of 8-O-acetylganoside methyl ester is 100 μM-200 μM.

[0022] The breast cancer cell inhibitor of the present invention contains an active ingredient, 8-O-acetylganoside methyl ester (Barlerin), which can effectively inhibit the cell viability, proliferation, migration and invasion, and colony formation of breast cancer cells. Furthermore, it exerts its tumor-suppressing effect by inhibiting the EGFR (epidermal growth factor receptor) related signaling pathway, thereby inhibiting the expression of oncogenic proteins HIF-1α and c-Myc, achieving comprehensive and effective inhibition of breast cancer cells.

[0023] Preferably, the breast cancer cells are triple-negative breast cancer cells.

[0024] Compared with other breast cancer cells, triple-negative breast cancer cells not only have a stronger cell proliferation index and migration ability, but also have a stronger invasiveness and clonogenic ability, which makes them less effective against conventional inhibitors.

[0025] The present invention has found through experiments that the active ingredient 8-O-acetylganoside methyl ester not only has a good inhibitory effect on the proliferation and colony formation of triple-negative breast cancer cells, but also effectively inhibits their migration and invasion capabilities. The inhibitory mechanism is that Barlerin targets the epidermal growth factor receptor (EGFR) for breast cancer cells. This result indicates that it can provide a low-toxicity and high-efficiency natural treatment candidate for triple-negative breast cancer, a subtype lacking targeted drugs, effectively overcoming the drug resistance problem of existing EGFR inhibitors and enhancing chemotherapy sensitivity. It has greater significance in breast cancer-related research and clinical applications.

[0026] The breast cancer cell inhibitors described above in this invention can be used to prepare drugs for treating breast cancer.

[0027] Preferably, the drug also includes other pharmaceutically acceptable excipients, said excipients including at least one of diluents, binders, absorbents, disintegrants, dispersants, humectants, solubilizers, buffers, and surfactants.

[0028] Preferably, the drug is one or more of the following formulations: tablet, powder, and liquid.

[0029] The present invention will be illustrated by specific embodiments below.

[0030] Example 1 Cell viability detection The triple-negative human breast cancer cell line used in this embodiment is MDA-MB-231 cells, which were purchased from Nanjing Shenghang Biotechnology Co., Ltd. The cell culture medium is DMEM medium containing 10% FBS and 1% penicillin and streptomycin.

[0031] In this embodiment, the cells were first cultured in an incubator at 37°C and 5% CO2.

[0032] The cultured MDA-MB-231 cells were then subjected to a 4×10⁻⁶ 3 / wells were seeded into 96-well plates, and 0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 150 μM, and 200 μM Barlerin (dissolved in cell culture medium containing 0.1% dimethyl sulfoxide (DMSO)) were added respectively. The plates were incubated at 37°C in a 5% CO2 incubator for 48 h. After that, the medium was replaced with fresh medium, and CCK8 cell proliferation and toxicity assay reagent was added. The plates were incubated for another h, and the absorbance was measured at 450 nm using a microplate reader. The experiment was repeated three times.

[0033] like Figure 1The CCK8 assay results showed that different concentrations of Barlerin significantly inhibited the activity of the triple-negative human breast cancer cell line MDA-MB-231. Specifically, cell activity decreased significantly at a Barlerin concentration of 50 μM; the inhibition was most pronounced at a concentration of 200 μM, with relative cell activity decreasing to 66.03 ± 3.92% (P < 0.0001), indicating that Barlerin can effectively inhibit the activity of triple-negative breast cancer cells.

[0034] Example 2 EdU cell proliferation detection MDA-MB-231 cells were loaded at 2×10 5 / wells were seeded into 6-well plates, and 0 μM, 100 μM, 150 μM, and 200 μM Barlerin (dissolved in cell culture medium containing 0.1% dimethyl sulfoxide (DMSO)) were added respectively. The plates were then incubated in a 5% CO2 incubator at 37°C for 48 h. Half of the original culture medium was discarded, and an equal volume of 2× EdU working solution (diluted with fresh culture medium) was added. The plates were then incubated in a 5% CO2 incubator at 37°C for another 2 h.

[0035] The culture medium was removed, and the cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Then, 500 mL of Click reaction solution was added, and the cells were incubated at room temperature in the dark for 30 min. After washing, 1 mL of Hoechst 33342 staining solution was added for nuclear staining for 10 min. Cell proliferation was recorded under an inverted fluorescence microscope. EdU-positive cells were considered proliferating cells, and Hoechst-positive cells were considered total cells. The ratio of EdU-positive cells to Hoechst-positive cells was the proliferation rate. The experiment was repeated three times.

[0036] like Figure 2 As shown in the results of the EdU cell proliferation assay kit, different concentrations of Barlerin significantly inhibited the proliferation of the triple-negative human breast cancer cell line MDA-MB-231.

[0037] When the Barlerin concentration reached 100 μM, the proportion of EdU-positive cells significantly decreased; when the Barlerin concentration reached 200 μM, the proportion of EdU-positive cells reached its lowest point, decreasing from 36.54 ± 2.52% to 23.32 ± 0.74% (P < 0.0001). Figure 2 (A and B in the original text). This result indicates that Barlerin can significantly inhibit the proliferation of triple-negative breast cancer cells.

[0038] Example 3 Cell Migration Experiment MDA-MB-231 cells were resuspended in serum-free DMEM medium at a concentration of 5 × 10⁻⁶ cells / mL. 5 Cells per mL were seeded, and 100 μL of cell suspension was seeded into the upper layer of an 8 μm pore size Transwell chamber. 600 μL of medium containing 10% FBS was added to the lower layer, and 0 μM, 100 μM, 150 μM, and 200 μM Barlerin (dissolved in cell culture medium containing 0.1% dimethyl sulfoxide (DMSO)) were added to the lower layer medium, respectively. The cells were incubated for 48 h in a 5% CO2 incubator at 37°C. The cells in the upper layer of the chamber were carefully wiped off with a cotton swab and washed several times with PBS. The chambers were then fixed in 4% paraformaldehyde for 15 min, stained with 1% crystal violet for 15 min, washed with PBS, and the number of cells that penetrated the filter membrane was recorded under an inverted microscope. The experiment was repeated three times.

[0039] according to Figure 3 The experimental results show that when the Barlerin concentration reaches 100 μM, the number of cells that can cross the filter membrane decreases significantly. When the Barlerin concentration reaches 200 μM, the number of cells that can cross the filter membrane reaches its lowest point, decreasing from 430.33 ± 10.07 cells to 208.33 ± 17.16 cells (P < 0.0001). Figure 3 (A and B in the original text). This result indicates that Barlerin significantly inhibits the migration ability of triple-negative breast cancer cells.

[0040] Example 4 Cell Invasion Experiment After mixing the matrix gel and serum-free culture medium at a 1:4 ratio, 40 μL of the diluted matrix gel was spread evenly on the upper layer of an 8 μm pore size Transwell chamber and incubated at 37°C for 30 min. MDA-MB-231 cells were then resuspended in serum-free DMEM medium at a cell concentration of 8 × 10⁻⁶ cells / mL. 5 Cells per mL were seeded, and 100 μL of cell suspension was seeded into the upper layer of a Transwell chamber pre-coated with matrix gel. 600 μL of medium containing 10% FBS was added to the lower layer of the chamber, and 0 μM, 100 μM, 150 μM, and 200 μM Barlerin (dissolved in cell culture medium containing 0.1% dimethyl sulfoxide (DMSO)) were added to the lower layer of medium, respectively. The chambers were incubated in a 5% CO2 incubator at 37°C for 48 h. The matrix gel and cells in the upper layer of the chamber were carefully wiped off with a cotton swab, and the chambers were washed several times with PBS. The chambers were then fixed in 4% paraformaldehyde fixative for 15 min, stained with 1% crystal violet for 15 min, washed with PBS, and the number of cells that penetrated the filter membrane was recorded under an inverted microscope. The experiment was repeated three times.

[0041] according to Figure 4The results showed that when the Barlerin concentration reached 100 μM, the number of cells that passed through the matrix gel and filter membrane decreased significantly. When the Barlerin concentration reached 200 μM, the number of cells that passed through the matrix gel and filter membrane decreased to the lowest level, from 1514.00 ± 232.42 cells to 426.67 ± 19.50 cells (P < 0.0001). Figure 4 (A and B in the original text). This result indicates that Barlerin significantly inhibits the invasive ability of triple-negative breast cancer cells.

[0042] Example 5: Cloning Experiment MDA-MB-231 cells were fed at a rate of 1×10⁻⁶. 3 Cells were seeded in 6-well plates, and 0 μM, 100 μM, 150 μM, and 200 μM Barlerin solutions (dissolved in cell culture medium containing 0.1% dimethyl sulfoxide (DMSO)) were added to each well. Cells were incubated in a 5% CO2 incubator at 37°C, with the culture medium changed every 3 days for 15 days to allow cell colonies to form. Cells were fixed with 4% paraformaldehyde for 15 min, stained with 1% crystal violet for 15 min, washed with PBS, and air-dried at room temperature. Cell colonies with ≥40 cells were counted. The experiment was repeated three times.

[0043] according to Figure 5 The results of the colony formation assay showed that different concentrations of Barlerin significantly inhibited the colony formation ability of the triple-negative human breast cancer cell line MDA-MB-231. Specifically, at a Barlerin concentration of 100 μM, the number of cell colonies formed was significantly reduced, and the cell colonies were also smaller. When the Barlerin concentration reached 200 μM, cell activity was most significantly inhibited, with the number of cell colonies decreasing from 149.00±16.64 to 97.00±8.89% (P<0.001). Figure 5 (A and B) indicates that Barlerin can effectively inhibit the clonogenic ability of triple-negative breast cancer cells.

[0044] Example 6 Network Pharmacology Analysis This embodiment utilizes the PubChem database to determine the chemical structure of Barlerin and obtain the compound's SMILES code, as detailed below: CC(=O)O[C@]1(C[C@H]([C@H]2[C@@H]1[C@@H](OC=C2C(=O)OC)O[C@H]3[C@@H]([C@H]([C@@H]([C@H](O3)CO)O)O)O)O)C.

[0045] The obtained drug SMILES were input into the Swiss Target database for prediction to obtain potential target genes for Barlerin. Potential target genes for breast cancer were then searched in the Genecards and OMIM databases. The obtained Barlerin and breast cancer targets were input into Venny 2.1.0 (https: / / bioinfogp.cnb.csic.es / tools / venny / ) to determine their intersection and a Venn diagram of Barlerin and breast cancer was plotted.

[0046] By searching for Barlerin-potential target genes in breast cancer on the String website, a network diagram can be obtained. Based on the results of Degree, Closeness, and Betweenness analysis of the network diagram using Cytoscape software, a new network diagram can be generated, the core targets can be obtained, and the results of drug-disease target protein interactions can be output.

[0047] The selected core targets were subjected to GO functional enrichment analysis and KEGG pathway enrichment analysis using Metascape (https: / / metascape.org / ).

[0048] Disease-related targets were obtained from the OMIM and Genecards databases, and standard targets were predicted using the Swiss TargetPrediction online database.

[0049] Using Venny 2.1.0, the intersection of the Barlerin target and the breast cancer target was calculated, resulting in 6 genes ( Figure 6 A). These six intersecting genes were input into the STRING database for PPI analysis, revealing the core targets as follows: EGFR, HRAS, FGFR1, SRC, MMP2, and AURKA (…). Figure 6 B). GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on the selected core targets using the Metascape online database.

[0050] GO functional enrichment analysis showed that in breast cancer, Barlerin is mainly involved in the eph receptor signaling pathway, glutamatergic synapses, histone H3Y41 kinase activity, EGFR signaling pathway, etc. Figure 6 C).

[0051] KEGG pathway analysis revealed that in breast cancer, Barlerin is primarily involved in the gonadotropin-releasing hormone signaling pathway, endocrine resistance, relaxin signaling pathway, estrogen signaling pathway, Rap1 signaling pathway, regulation of the actin cytoskeleton, and resistance to EGFR tyrosine kinase inhibitors. Figure 6 D).

[0052] Example 7: Western blot experiment MDA-MB-231 cells were loaded at 2×10 5 / wells were seeded into 6-well plates, and 0 μM, 100 μM, 150 μM, and 200 μM Barlerin (dissolved in cell culture medium containing 0.1% dimethyl sulfoxide (DMSO)) were added respectively. Cells were incubated in a 5% CO2 incubator at 37°C for 48 h. Cells were then collected, washed twice with PBS, and subjected to cell lysis buffer containing 1 mM PMSF. Cells were sonicated on ice (3 s, 3 s pause, repeated 4 times), and incubated on ice for 20 min. After centrifugation at 12000 rpm for 15 min at 4°C, the supernatant was transferred to a new EP tube. Protein concentration was determined using a BCA assay kit. Remaining protein was added to 5× protein loading buffer and denatured in a 100°C metal bath for 10 min. 15 μg of each sample was added to a 12% precast gel, electrophoresed at 150 V for 1 h, followed by electroporation at 300 mA for 2 h. The protein was then transferred to an NC membrane. The membrane was then placed in a 5% CO2 incubator. The membrane was blocked in BSA with TBS for 2 hours, then incubated with primary antibody at 4°C overnight. The membrane was washed with TBST three times for 5 minutes each time, incubated with fluorescent secondary antibody at room temperature for 1 hour, and washed with TBST three times for 5 minutes each time. The membrane was then developed using a fluorescence imaging system, and the grayscale values ​​of the bands were analyzed using ImageJ software. The experiment was repeated three times. The experimental results are as follows: Figure 7 As shown.

[0053] according to Figure 7 The experimental results showed that Barlerin significantly inhibited the expression of EGFR protein in the triple-negative human breast cancer cell line MDA-MB-231. When the Barlerin concentration reached 150 μM, EGFR protein expression was significantly inhibited; the inhibition was most pronounced at a Barlerin concentration of 200 μM, with a relative expression level decreasing to 0.83 ± 0.06 (P < 0.01). Figure 7 AB).

[0054] Meanwhile, this embodiment also detected the expression of the oncogenic proteins HIF-1α and c-Myc. When the Barlerin concentration reached 100 μM, the expression of c-Myc protein was significantly reduced; when the Barlerin concentration reached 150 μM, the expression of HIF-1α protein was also significantly reduced; and when the Barlerin concentration reached 200 μM, the expression of both HIF-1α and c-Myc proteins decreased to the lowest level, with relative expression levels of 0.84±0.04 (P<0.01) and 0.66±0.03 (P<0.0001), respectively. Figure 7 A, CD).

[0055] The above experimental results indicate that Barlerin's target for breast cancer is most likely EGFR, and it can effectively promote the expression of oncogenes in breast cancer cells.

[0056] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A breast cancer cell inhibitor comprising 8-O-acetylrubusoside methyl ester, characterized in that, The concentration of the 8-O-acetylrubroglycoside methyl ester is 100 μM-200 μM.

2. The breast cancer cell inhibitor containing 8-O-acetylmethylshanzhiside A according to claim 1, characterized in that, The solvent of the inhibitor is a cell culture medium containing 0.1% dimethyl sulfoxide.

3. Use of the breast cancer cell inhibitor containing 8-O-acetylrubroglycoside methyl ester according to claim 1 or 2 in the preparation of a medicament for treating breast cancer.

4. Use of the breast cancer cell inhibitor containing 8-O-acetylrubusoside methyl ester according to claim 3 for the preparation of a medicament for the treatment of breast cancer, characterized in that, The breast cancer cells are triple negative breast cancer cells.

5. Use of the breast cancer cell inhibitor containing 8-O-acetylrubusoside methyl ester according to claim 4 for the preparation of a medicament for the treatment of breast cancer, characterized in that, The triple negative breast cancer cells are MDA-MB-231 cells.

6. A medicament for the preparation of a medicament for the treatment of breast cancer, characterized in that, The medicament comprises the breast cancer cell inhibitor according to claim 1 or 2.

7. A medicament for the preparation of a medicament for the treatment of breast cancer according to claim 6, characterized in that, The medicament further comprises other pharmaceutically acceptable excipients, which include at least one of diluents, binders, absorbents, disintegrants, dispersants, wetting agents, co-solvents, buffers and surfactants.

8. A medicament for the preparation of a medicament for the treatment of breast cancer according to claim 6, characterized in that, The preparation of the medicament is one or more of tablets, powders, liquid preparations.

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