Use of berberine hydrochloride in the preparation of an inhibitor of HMGB3

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a novel use of berberine hydrochloride and a pharmaceutical composition. Berberine hydrochloride has the structure shown below, and it significantly inhibits the expression of HMGB3 in triple-negative breast cancer cells, significantly inhibits the proliferation, colony formation, invasion, and migration of triple-negative breast cancer cells, and promotes apoptosis in triple-negative breast cancer cells. Therefore, berberine hydrochloride can be used to prepare drugs for the prevention and / or treatment of triple-negative breast cancer. Furthermore, berberine hydrochloride can enhance the drug sensitivity of cisplatin; the combined use of berberine hydrochloride and cisplatin has a strong synergistic effect in the treatment of triple-negative breast cancer, providing more treatment options for clinical treatment of triple-negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a new use of berberine hydrochloride and a pharmaceutical composition. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, seriously threatening women's health. Triple-negative breast cancer (TNBC), as a highly malignant subtype of breast cancer with a poor prognosis, has seen slow progress in treatment in recent years due to the lack of clear therapeutic targets in the past. Recurrent or metastatic TNBC typically has a poor prognosis, with a 5-year survival rate of less than 15%, significantly lower than the overall 5-year survival rate for breast cancer patients (31%). The main goals of its treatment are to slow disease progression, prolong survival time, and improve patients' quality of life. Especially for advanced TNBC, treatment options are relatively limited, and further optimization of treatment recommendations is needed. Currently, treatment methods for TNBC include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, recurrence and drug resistance are also important reasons why TNBC is difficult to cure. Enhancing the efficacy of cisplatin remains a challenge for clinicians.

[0003] Triple-negative breast cancer (TNBC) presents significant challenges due to the lack of specific endocrine therapy targets and HER2-targeted therapy. Cisplatin, a platinum-based chemotherapy drug, is commonly used to treat advanced TNBC, especially in patients with BRCA mutations. Although cisplatin is effective in some patients, the development of resistance significantly limits its clinical application.

[0004] HMGB3 (High Mobility Group Box 3) is a small non-histone DNA-binding protein belonging to the High Mobility Group Box (HMGB) family. This family includes HMGB1, HMGB2, and HMGB3, whose different functions and expression patterns in cells make them crucial in biological research. HMGB3 plays an important role in regulating cell cycle, DNA repair, and transcriptional regulation. HMGB3 is highly expressed in various tumors and is closely related to tumor occurrence, development, and treatment resistance. Therefore, researchers are dedicated to exploring the mechanism of action of HMGB3 in tumors and developing anti-tumor therapeutic strategies targeting HMGB3. Our research found that HMGB3 is highly expressed in triple-negative breast cancer (TNC) tissues and is associated with poor prognosis in TNC patients. Therefore, HMGB3 may be a potential therapeutic target for TNC. Based on this, our further research found that the compound berberine hydrochloride can effectively inhibit HMGB3 activity to exert an anti-tumor effect and can also enhance the sensitivity of TNC to platinum-based drugs, thus enabling its use in the preparation of anti-tumor drugs. Summary of the Invention

[0005] The purpose of this invention is to provide a novel use for berberine hydrochloride and a pharmaceutical composition in which berberine hydrochloride significantly inhibits the expression of HMGB3 in triple-negative breast cancer cells, significantly inhibits the proliferation, colony formation, invasion, and migration of triple-negative breast cancer cells, and promotes apoptosis of triple-negative breast cancer cells. Furthermore, berberine hydrochloride can enhance the drug sensitivity of cisplatin. The combined use of berberine hydrochloride and cisplatin has a synergistic effect in the treatment of triple-negative breast cancer.

[0006] Specifically, the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides the use of berberine hydrochloride in the preparation of a medicament for the prevention and / or treatment of triple-negative breast cancer, said berberine hydrochloride having the molecular formula C0. 19 H 14 ClNO4, the chemical structural formula of which is shown below:

[0008]

[0009] In one embodiment, in the above-described use, the berberine hydrochloride prevents and / or treats triple-negative breast cancer by inhibiting the expression of HMGB3.

[0010] In one embodiment, in the above-described use, the berberine hydrochloride prevents and / or treats triple-negative breast cancer by inhibiting the proliferation, invasion, and migration of triple-negative breast cancer cells and promoting apoptosis of triple-negative breast cancer cells.

[0011] In one embodiment, in the above-described use, the berberine hydrochloride can be prepared into an oral formulation with a pharmaceutically acceptable carrier.

[0012] In one embodiment, the oral formulation is a tablet, capsule, granule, or oral liquid.

[0013] In a second aspect, the present invention provides the use of the above-mentioned berberine hydrochloride in the preparation of HMGB3 inhibitors.

[0014] In one embodiment, in the above-described uses, the HMGB3 inhibitor is used for non-diagnostic or therapeutic purposes.

[0015] Preferably, the HMGB3 inhibitor is used for in vitro scientific research purposes.

[0016] In a third aspect, the present invention provides the use of the above-mentioned berberine hydrochloride in the preparation of drugs that inhibit or knock out HMGB3.

[0017] In a fourth aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of triple-negative breast cancer, the pharmaceutical composition comprising cisplatin and berberine hydrochloride in a molar ratio of 1-4:1.

[0018] In one embodiment, the molar ratio of cisplatin to berberine hydrochloride in the above pharmaceutical composition is 1.5-4:1, preferably 4:1.

[0019] In a fifth aspect, the present invention provides the use of the pharmaceutical composition described in the fourth aspect above in the preparation of a medicament for the prevention and / or treatment of triple-negative breast cancer.

[0020] In one embodiment, in the above-described use, the cisplatin and the berberine hydrochloride are prepared into an oral formulation with a pharmaceutically acceptable carrier.

[0021] In one embodiment, the oral formulation is a tablet, capsule, granule, or oral liquid.

[0022] As used herein, the term "pharmaceutically acceptable carrier" in this invention refers to a conventional pharmaceutical carrier in the field of pharmaceutical formulations, selected from one or more of fillers, binders, lubricants, suspending agents, disintegrants, wetting agents, flavoring agents, pigments, solvents, and surfactants.

[0023] The fillers of this invention include, but are not limited to, starch, sucrose, dextrin, microcrystalline cellulose, lactose, glucose, etc.; the binders include, but are not limited to, water, ethanol, syrup, starch paste, sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, etc.; the lubricants include, but are not limited to, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the suspending agents include, but are not limited to, polysaccharides such as farnesian gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan, etc.; the disintegrants include, but are not limited to, starch effervescent mixtures such as sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; and the solvents include, but are not limited to, water, balanced salt solutions, etc.

[0024] The above-mentioned formulations can be prepared using conventional processes in the field of pharmaceutical formulation.

[0025] As used herein, the berberine hydrochloride and cisplatin of this invention are commercially available products.

[0026] In the pharmaceutical applications described above, the timing, frequency, and duration of administration of various active ingredients need to be determined based on the specific diagnostic results of the condition, which is within the technical scope of those skilled in the art.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) This invention provides a novel use for berberine hydrochloride, which significantly inhibits the expression of HMGB3 in triple-negative breast cancer cells, significantly inhibits the proliferation, colony formation, invasion, and migration of triple-negative breast cancer cells, and promotes apoptosis of triple-negative breast cancer cells. Therefore, berberine hydrochloride can be used to prepare drugs for the prevention and / or treatment of triple-negative breast cancer.

[0029] (2) The berberine hydrochloride provided by the present invention can also enhance the drug sensitivity of cisplatin. The combined use of berberine hydrochloride and cisplatin has a strong synergistic effect in the treatment of triple-negative breast cancer, providing more treatment options for the clinical treatment of triple-negative breast cancer. Attached Figure Description

[0030] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings, "*" represents P < 0.05, indicating statistical significance between groups.

[0031] Figure 1 This shows the expression of HMGB3 in triple-negative breast cancer tissues and triple-negative breast cancer cell lines. Figure 1 In the above, A: The expression level of HMGB3 in breast cancer tumor tissues and normal tissues was analyzed using the GEPIA2 online database; B: The expression level of HMGB3 in different subtypes of breast cancer was analyzed using the GEO dataset GSE45827; C: The survival prognosis of patients with triple-negative breast cancer who expressed high and low HMGB3 was analyzed using GSE58812; D: The expression level of HMGB3 in different subtypes of breast cancer was analyzed by immunohistochemistry; E: The expression of HMGB3 in different subtypes of breast cancer was detected by Western blot experiment; F and G: HMGB3 knockdown and overexpression cell lines were constructed and validated by Western blot experiment.

[0032] Figure 2 The results showed that HMGB3 can promote the proliferation of triple-negative breast cancer cells and inhibit the apoptosis of triple-negative breast cancer cells. Figure 2 In the figure, AD: EdU cell proliferation assay to detect the effect of HMGB3 on the proliferation ability of triple-negative breast cancer cells; E: mouse tumor size graph; F: mouse tumor growth curve; G: mouse tumor weight statistics graph.

[0033] Figure 3 HMGB3 was shown to promote the migration and invasion of triple-negative breast cancer cells. Figure 3 In the diagram, A: Western blot assay was used to detect the effect of HMGB3 on the migration and invasion ability of triple-negative breast cancer cells; B and C: Transwell assays were used to detect the effect of HMGB3 on the migration and invasion ability of triple-negative breast cancer cells.

[0034] Figure 4 HMGB3 was shown to reduce cisplatin-mediated antitumor activity. Figure 4 In the study, A: an HMGB3 knockdown cell model was constructed in the BT549 cell line, and transcriptome sequencing and pathway enrichment analysis were performed; B: the effect of HMGB3 on the sensitivity of triple-negative breast cancer cells to cisplatin was detected by CCK-8 assay.

[0035] Figure 5 HMGB3 was shown to reduce cisplatin-mediated antitumor activity. Figure 5 In the diagram, A: Comet assays confirm that knocking down HMGB3 promotes DNA damage in triple-negative breast cancer cells induced by cisplatin. B: CCK-8 assays confirm that knocking down HMGB3 increases the sensitivity of triple-negative breast cancer cells to cisplatin.

[0036] Figure 6 The results showed that berberine hydrochloride can inhibit the protein expression level of HMGB3. Figure 6 In this study, Western blot analysis was used to detect the expression of HMGB3 in triple-negative breast cancer cells after treatment with different concentrations of berberine hydrochloride.

[0037] Figure 7 Studies have shown that berberine hydrochloride can inhibit the proliferation, migration, and invasion of triple-negative breast cancer cells and promote apoptosis of triple-negative breast cancer cells. Figure 7 In the study, A: CCK-8 assay was used to detect the proliferation of triple-negative breast cancer cells after treatment with different concentrations and durations of berberine hydrochloride; B and C: Berberine hydrochloride can inhibit the invasive formation ability of triple-negative breast cancer cells; D: CCK-8 assay was used to detect the inhibitory effect of different concentrations of berberine hydrochloride combined with cisplatin on cells.

[0038] Figure 8 The results showed that berberine hydrochloride can enhance cisplatin-mediated antitumor activity. Figure 8 In the diagram, A: CCK-8 assay to detect cell proliferation of HCC1937 and BT549 cells after cisplatin combined with berberine hydrochloride; B: Transwell assay to detect cell invasion after cisplatin combined with berberine hydrochloride; C: Comet assay to verify DNA damage in cells after cisplatin combined with berberine hydrochloride; D: Endpoint graph of mouse tumor size in xenograft model experiment; E: Statistical graph of mouse tumor weight; F: Curve of mouse tumor volume change. Detailed Implementation

[0039] The embodiments of the present invention are described in detail below. The embodiments are provided to better illustrate the content of the present invention and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0040] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0041] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0042] Example 1

[0043] Pharmacodynamic experiments

[0044] 1. Experimental Methods

[0045] 1.1 Cell Proliferation Assays: CCK-8 Assay: Triple-negative breast cancer cells were seeded in 96-well plates and treated with different concentrations (0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM) of berberine hydrochloride. After culturing for 24, 48, and 72 h, 10 μl of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2 h. The absorbance at 450 nm was measured to assess cell proliferation. EdU Cell Proliferation Assay: Triple-negative breast cancer cells were seeded in 96-well plates. After cell attachment, EdU fluorescent labeling agent was added to the cell culture medium and incubated at 37°C for 4 h. The cells were then fixed with 4% paraformaldehyde for 20 min. The fixed cells were permeabilized for 10 min to allow the fluorescent labeling agent to penetrate the cell membrane. The EdU detection kit was used according to the manufacturer's instructions. Finally, the fluorescence signal of the cells was observed using a fluorescence microscope to determine the intracellular EdU uptake and thus assess cell proliferation activity.

[0046] 1.2 Transwell Assay: Prepare 24-well plates, add 500 μl of complete culture medium to each well, and then place a Transwell chamber inside. The chamber for the invasion assay contains matrix gel, while the chamber for the migration assay does not. Based on the migration and invasion characteristics of different cells, culture in serum-free medium at 37°C for several hours to several days. After culture, remove the chambers, discard the culture medium, wash with PBS, fix with 4% paraformaldehyde for 20 minutes, and stain with crystal violet for 20 minutes. After staining, scrape off the upper layer of cells from the chamber with a cotton swab, clean thoroughly, air dry at room temperature, photograph, and count. The migration and invasion abilities of cells are assessed by analyzing the number and morphological characteristics of migrating or invading cells.

[0047] 1.3 In vivo tumor-bearing experiment in nude mice: The Balb / c Nude mice used in this experiment were 4-6 week old female mice. HCC1937 triple-negative breast cancer cells (5×10⁻⁶) were introduced into the mice. 6A 100 μl suspension of cells was prepared and injected into the back of nude mice to complete the tumor-bearing experiment. After tumor formation, the mouse weight and tumor size were measured periodically. The tumor volume was calculated using the formula: length × width × width × 0.5. When the tumor reached a suitable size, the nude mice were sacrificed, the tumor was removed, weighed, and photographed. Some tumors were fixed in paraformaldehyde and embedded in paraffin, while the remaining tumors were preserved in liquid nitrogen for protein extraction.

[0048] 1.4 Western blot assay: Triple-negative breast cancer cells were collected, lysed with RIPA buffer, and protease and phosphatase inhibitors were added. The cells were centrifuged at 12,000 rpm for 30 minutes at 4°C to obtain the protein supernatant. Protein concentration was determined using the BCA method. Protein samples were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was then blocked and incubated with antibodies. Finally, protein expression was observed under optical conditions by adding a chromogenic substrate. These steps allow for the detection of the target protein's expression level in the sample and enables semi-quantitative or quantitative analysis.

[0049] 1.5 Comet Assay: Collect cells, centrifuge, remove the culture medium, and resuspend the cells in PBS. Dilute the cells 1×10⁻⁶. 6 Cells / mL. Mix the cell suspension with low-melting-point agarose and rapidly add the mixture dropwise onto a pre-cooled glass slide to form a thin layer, allowing it to solidify. Place the slide in an electrophoresis tank, pour in electrophoresis buffer, and apply a voltage of 25V for 30 minutes. Stain with a fluorescent dye to visualize the DNA. Wash with PBS after staining to remove unbound dye. Observe the slide using a fluorescence microscope to assess DNA damage. Quantify the degree of DNA damage by counting the number and morphology of "comets".

[0050] 1.6 Immunohistochemistry: Immunohistochemistry was used to detect HMGB3 expression in different subtypes of breast cancer. Tissue samples were fixed on glass slides and dehydrated and dewaxed to maintain structural integrity. Antibodies were used to detect specific antigen proteins. Target proteins were labeled with staining agents to make them visible under a microscope. The stained samples were observed under a microscope to determine the presence and distribution of the target proteins. Immunohistochemical staining was scored according to the following criteria: "-" indicates no positive cells, "+" indicates 1-20% positive cells, "++" indicates 20-50% positive cells, and "+++" indicates 50-100% positive cells. IHC expression of the protein was evaluated: "- to +" defined as low expression; "++ to +++" defined as high expression.

[0051] 2. Experimental Results

[0052] 2.1 Expression of HMGB3 in triple-negative breast cancer tissues and cell lines. Figure 1 As shown in Figure A, to analyze the expression of HMGB3 in breast cancer and normal tissues, the GEPIA2 online database was used. Compared with normal breast cancer tissue, the mRNA expression level of HMGB3 in breast cancer tissue was significantly increased (P<0.05). Figure 1 As shown in Figure B, to investigate the expression of HMGB3 in different subtypes of breast cancer, bioinformatics analysis using the GEO public dataset (GSE45827) revealed that triple-negative breast cancer is associated with higher levels of HMGB3 expression. Meanwhile, from... Figure 1 As shown in Figure C, in order to analyze the relationship between HMGB3 and the prognosis of triple-negative breast cancer patients, bioinformatics analysis using the GEO public dataset (GSE58812) revealed that triple-negative breast cancer patients with high expression of HMGB3 had a poorer prognosis.

[0053] To further verify the differences in HMGB3 expression in different subtypes of breast cancer, by Figure 1 As shown in the data from the study, immunohistochemical analysis of HMGB3 expression in different subtypes of breast cancer tissues revealed that triple-negative breast cancer tissues exhibited higher HMGB3 expression levels. Figure 1 As shown in the results, Western blot experiments revealed that HMGB3 protein expression levels were significantly higher in triple-negative breast cancer cell lines HCC1937 and BT549. Figure 1 As shown in F and G, HMGB3 knockdown and overexpression (HCC1937 and BT549 cells) cell lines were constructed using lentiviruses, and the above HMGB3 expression results were verified by Western blot experiments.

[0054] 2.2 HMGB3 promotes the proliferation of triple-negative breast cancer cells and inhibits their apoptosis. To explore whether HMGB3 can affect the proliferation ability of triple-negative breast cancer cells, an EdU assay was performed to detect cell proliferation. Figure 2 According to the results of the EdU experiment, overexpression of HMGB3 promoted the growth and proliferation of HCC1937 and BT549 cells compared with the control group; while knockdown of HMGB3 significantly inhibited the growth and proliferation of HCC1937 and BT549 cells. Figure 2 As can be seen from the data in the experiment, the Balb / c Nude mice used were 4-6 week old female mice. The HCC1937 (Ctrl, Sh-HMGB3#1, Sh-HMGB3#2) triple-negative breast cancer cells (5 × 10⁻⁶) were constructed. 6(Each cell) was prepared into a 100 μl suspension and divided into three groups, which were injected into the backs of nude mice. One week after tumor formation, the mice's weight and tumor size were measured periodically. The tumor volume was calculated using the formula: length × width × width × 0.5. Three weeks after administration, the nude mice were sacrificed, and the tumors were removed, weighed, and photographed. Figure 2 As shown in F and G, knockdown of HMGB3 expression significantly inhibited the growth of subcutaneous xenografts in mice.

[0055] 2.3 HMGB3 promotes cell migration and invasion. The potential effects of HMGB3 on the migration and invasion of triple-negative breast cancer cells were further investigated. Figure 3 As shown in Figure A, Western blot experiments revealed that knocking out HMGB3 significantly inhibited the migration and invasion abilities of triple-negative breast cancer cells HCC1937 and BT549; while overexpression of HMGB3 significantly enhanced the migration and invasion abilities of HCC1937 and BT549 cells. Figure 3 As shown in Figures B and C, Transwell experiments revealed that knocking out HMGB3 significantly inhibited the migration and invasion abilities of triple-negative breast cancer cells HCC1937 and BT549; while overexpression of HMGB3 significantly enhanced the migration and invasion abilities of triple-negative breast cancer cells HCC1937 and BT549.

[0056] 2.4 HMGB3 can reduce cisplatin-mediated antitumor activity. Next, to further explore the role of HMGB3 in triple-negative breast cancer, an HMGB3 knockdown cell line was constructed using BT549 triple-negative breast cancer cells, and transcriptome sequencing analysis was performed. Figure 4 As shown in Figure A, HMGB3 affects many biological functions of triple-negative breast cancer cells, such as DNA damage, glycolysis, apoptosis, and platinum resistance. Triple-negative breast cancer cells typically exhibit good sensitivity to cisplatin in the early stages of treatment, mainly due to their defective DNA damage repair mechanisms. Advanced triple-negative breast cancer patients often develop cisplatin resistance. Figure 4 As shown in Figure B, the CCK-8 assay demonstrated that cisplatin promotes apoptosis in triple-negative breast cancer cells HCC1937 and BT549, and the inhibitory effect increases with increasing concentration.

[0057] 2.5HMGB3 attenuated cisplatin-mediated activity against triple-negative breast cancer. Figure 5 The comet assay revealed that, compared to the control group, knocking down HMGB3 significantly increased DNA damage in triple-negative breast cancer cells treated with cisplatin. This suggests that knocking out HMGB3 in vivo can improve the sensitivity of tumor cells to cisplatin treatment.

[0058] 2.6 Berberine hydrochloride inhibits HMGB3 expression. HCC1937 and BT549 triple-negative breast cancer cells were treated with different concentrations of berberine hydrochloride (0 μM, 10 μM, 20 μM, 40 μM, 80 μM) for 24 hours. Cells were then collected, proteins were extracted, and HMGB3 expression was detected using Western blot. Figure 6 It is known that berberine hydrochloride can inhibit the expression of HMGB3 in triple-negative breast cancer cells in a dose-dependent manner, with higher drug concentrations resulting in stronger inhibition of HMGB3 expression.

[0059] 2.7 Berberine hydrochloride inhibited the proliferation, migration, and invasion of triple-negative breast cancer cells and promoted their apoptosis. HCC1937 and BT549 cells were treated with different concentrations (0 μM, 10 μM, and 20 μM) of berberine hydrochloride. The growth and proliferation of HCC1937 and BT549 cells were detected using CCK-8 assay. Figure 7 As shown in Figure A, berberine hydrochloride significantly inhibited the survival rate of HCC1937 and BT549 cells in a dose- and time-dependent manner. The migration and invasion abilities of HCC1937 and BT549 cells were assessed using a Transwell assay. Figure 7 As shown in Figures B and C, berberine hydrochloride inhibits the migration and invasion abilities of HCC1937 and BT549 cells, and the inhibitory effect increases with increasing concentration. Figure 7 As shown in Figure D, the CCK-8 assay demonstrated that low-dose berberine hydrochloride combined with cisplatin significantly inhibited cell growth in triple-negative breast cancer.

[0060] 2.8 Berberine Hydrochloride Enhances the Sensitivity of Drug-Resistant Cell Lines to Cisplatin. To verify the effect of berberine hydrochloride on enhancing the sensitivity of triple-negative breast cancer cell lines HCC1937 and BT549 to cisplatin, experimental group 1 received different concentrations of cisplatin (5 μM, 10 μM, 20 μM, 30 μM); experimental group 2 received different concentrations of berberine hydrochloride (5 μM, 10 μM, 15 μM, 20 μM); and experimental group 3 received a combination of a fixed concentration (5 μM) of berberine hydrochloride and different concentrations of cisplatin (5 μM, 10 μM, 20 μM, 30 μM). Culture medium served as a blank control; 0.1% DMSO culture medium served as a control. After 72 hours of treatment, 10 μl of CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was then measured using a microplate reader. Cell proliferation survival rate = (OD value of drug-treated group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%, inhibition rate = 1 - survival rate. The combination index (CI) was calculated using CompuSyn software; a CI ≤ 0.9 was considered to indicate a potential synergistic effect between the two drugs. The results are shown in Table 1.

[0061] Table 1: Results of inhibition rate of triple-negative breast cancer cell proliferation by cisplatin, berberine hydrochloride, and their combination.

[0062]

[0063] Table 1 shows that when berberine hydrochloride is used alone, the inhibition rate of triple-negative breast cancer cell proliferation increases with increasing drug concentration. Furthermore, the combined use of berberine hydrochloride and cisplatin significantly increased the inhibition rate of triple-negative breast cancer cell proliferation compared to the single-drug group, indicating that the combined use of berberine hydrochloride and cisplatin has a strong synergistic effect in inhibiting the proliferation of triple-negative breast cancer cells.

[0064] Figure 8 In Figure A, tumor cell survival rate was determined using the drug concentrations listed in Table 1. The CCK-8 assay confirmed that the combined use of cisplatin and berberine hydrochloride enhanced the sensitivity of drug-resistant cell lines HCC1937 and BT549 to cisplatin. Transwell assays were used to detect the migration and invasion abilities of HCC1937 and BT549 cells, such as... Figure 8 As shown in Figure B, berberine hydrochloride combined with cisplatin inhibited the invasive ability of HCC1937 and BT549 cells. Figure 8 As shown in Figure C, comet assays confirmed that berberine hydrochloride combined with cisplatin significantly increased DNA damage in triple-negative breast cancer cell lines HCC1937 and BT549.

[0065] Next, the inventors evaluated the antitumor effect of berberine hydrochloride and cisplatin in combination using a nude mouse tumor-bearing experiment. The Balb / c Nude mice used were 4-6 week old females. HCC1937 triple-negative breast cancer cells (5 × 10⁻⁶) were introduced into the mice for the experiment. 6 A 100 μl suspension of cisplatin (containing 100 cells) was injected into the back of nude mice. One week after tumor formation, the mice were randomly divided into four groups: a control group, a cisplatin intraperitoneal injection group (25 mg / kg), a berberine hydrochloride intraperitoneal injection group (8 mg / kg), and a cisplatin and berberine hydrochloride combined group (cisplatin 25 mg / kg combined with berberine hydrochloride 8 mg / kg). Each group received treatment for 3 consecutive weeks. Mouse weight and tumor size were measured periodically. Tumor volume was calculated using the formula: length × width × width × 0.5. Three weeks after treatment, the nude mice were sacrificed, the tumors were removed, weighed, and photographed. Figure 8 As shown in the DF, the combined use of berberine hydrochloride and cisplatin can significantly inhibit tumor volume.

[0066] In summary, this invention found that berberine hydrochloride significantly inhibits the expression level of HMGB3 in triple-negative breast cancer cells, significantly suppresses the proliferation, colony formation, invasion, and migration abilities of triple-negative breast cancer cells, and promotes apoptosis in triple-negative breast cancer cells. Furthermore, berberine hydrochloride can enhance the drug sensitivity of cisplatin. The combined use of berberine hydrochloride and cisplatin has a strong synergistic effect in the treatment of triple-negative breast cancer, providing more treatment options for clinical treatment of triple-negative breast cancer.

[0067] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A pharmaceutical composition for the prevention or treatment of triple-negative breast cancer, characterized in that, The pharmaceutical composition comprises cisplatin and berberine hydrochloride, wherein the molar ratio of cisplatin to berberine hydrochloride is 1-4:1, and the berberine hydrochloride prevents or treats triple-negative breast cancer by inhibiting the expression of HMGB3.

2. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of cisplatin to berberine hydrochloride is 1.5-4:

1.

3. Use of the pharmaceutical composition of claim 1 or claim 2 in the preparation of a medicament for the prevention or treatment of triple-negative breast cancer.

4. The use according to claim 3, characterized in that, The cisplatin and berberine hydrochloride in the pharmaceutical composition are prepared into an oral formulation with a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that, The oral preparation is a tablet, capsule, granule, or oral liquid.

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