Application of coptisine hydrochloride in preparation of HMGB3 inhibitor

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a novel application of coptisine hydrochloride and a pharmaceutical composition. The coptisine hydrochloride has a structure shown in the specification, can significantly inhibit expression of HMGB3 in triple-negative breast cancer cells, can significantly inhibit proliferation, clone formation ability, invasion and migration ability of the triple-negative breast cancer cells, and can promote apoptosis of the triple-negative breast cancer cells. Therefore, the coptisine hydrochloride can be used for preparing the medicine for preventing and / or treating the triple negative breast cancer. And secondly, the coptisine hydrochloride can also enhance the drug sensitivity of the cis-platinum, and the coptisine hydrochloride and the cis-platinum are combined for use, so that a strong synergistic interaction effect is achieved in the aspect of treating the triple negative breast cancer, and more treatment choices are provided for clinically treating the triple negative breast cancer. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a new use of coptisine hydrochloride and a pharmaceutical composition. Background Art

[0002] Breast cancer is the most common malignant tumor in women worldwide, and it poses a serious threat to women's health. Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high malignancy and poor prognosis. Due to the lack of clear therapeutic targets in the past, treatment progress has been slow in recent years. Recurrent or metastatic triple-negative breast cancer usually has a poor prognosis, with a 5-year survival rate of less than 15%, which is significantly lower than the overall 5-year survival rate of breast cancer patients (31%). The main goal of its treatment is to delay disease progression, prolong survival time, and improve the quality of life of patients. Especially for the treatment of advanced triple-negative breast cancer, due to the relatively limited treatment options, further optimization of treatment recommendations is needed. At present, the treatment options for triple-negative breast cancer include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. However, recurrence and drug resistance are also one of the important reasons why triple-negative breast cancer is difficult to cure. How to enhance the efficacy of cisplatin has always been a difficult problem faced by clinicians.

[0003] Triple-negative breast cancer poses a great challenge due to its 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 drug resistance significantly limits its clinical application.

[0004] HMGB3 (High Mobility Group Box 3) is a small non-histone DNA binding protein that belongs to the high mobility group protein (HMGB) family. The members of this family include HMGB1, HMGB2 and HMGB3, etc. Their different functions and expression patterns in cells make them crucial in biological research. HMGB3 plays an important role in biological processes such as regulating cell cycle, DNA repair, and transcriptional regulation. HMGB3 is highly expressed in a variety of tumors and is closely related to the occurrence, development and treatment resistance of tumors. Therefore, researchers are committed to exploring the mechanism of action of HMGB3 in tumors and developing anti-tumor treatment strategies for HMGB3. The inventors found through research that HMGB3 is highly expressed in tissues of patients with triple-negative breast cancer and is associated with the poor prognosis of patients with triple-negative breast cancer. Therefore, HMGB3 may be a potential therapeutic target for triple-negative breast cancer. On this basis, the inventors further found that the compound coptisine hydrochloride can effectively inhibit the activity of HMGB3 to exert anti-tumor effects, and can enhance the drug sensitivity of triple-negative breast cancer to platinum to exert anti-tumor effects, so that it can be used to prepare anti-tumor drugs. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of coptisine hydrochloride and a pharmaceutical composition, wherein coptisine hydrochloride significantly inhibits the expression of HMGB3 in triple-negative breast cancer cells, significantly inhibits the proliferation, cloning ability, invasion and migration ability of triple-negative breast cancer cells, and promotes the apoptosis of triple-negative breast cancer cells. In addition, coptisine hydrochloride can also enhance the drug sensitivity of cisplatin, and the combination of coptisine hydrochloride and cisplatin has a synergistic effect in the treatment of triple-negative breast cancer.

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

[0007] In the first aspect, the present invention provides the use of coptisine hydrochloride in the preparation of a drug for preventing and / or treating triple-negative breast cancer, wherein the molecular formula of coptisine hydrochloride is C 19 H 14 ClNO 4 , the chemical structure is as follows:

[0008]

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

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

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

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

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

[0014] In one embodiment, in the above 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 use of the above-mentioned coptisine hydrochloride in the preparation of a drug for inhibiting or knocking out HMGB3.

[0017] In a fourth aspect, the present invention provides a pharmaceutical composition for preventing and / or treating triple-negative breast cancer, the pharmaceutical composition comprising cisplatin and coptisine hydrochloride, wherein the molar ratio of the cisplatin to the coptisine hydrochloride is 1-4:1.

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

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

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

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

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

[0023] The filler of the present invention includes but is not limited to starch, sucrose, dextrin, microcrystalline cellulose, lactose, glucose, etc.; the binder includes but is not limited to water, ethanol, syrup, starch slurry, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone, etc.; the lubricant includes but is not limited to stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the suspending agent includes but is not limited to polysaccharides such as acacia gum, agar, alginic acid, cellulose ether and carboxymethyl chitosan, etc.; the disintegrant includes but is not limited to starch effervescent mixture, i.e. sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the solvent includes but is not limited to water, a balanced salt solution, etc.

[0024] The above-mentioned various preparations can be prepared according to conventional techniques in the field of pharmaceutical preparations.

[0025] As used herein, coptisine hydrochloride and cisplatin of the present invention are purchased from commercially available products.

[0026] In the medical uses described above, the administration time, number of administrations and frequency of administration of various active ingredients need to be determined according to the specific diagnosis results of the disease, which is within the technical scope mastered by those skilled in the art.

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

[0028] (1) The present invention provides a new use of coptisine hydrochloride, which significantly inhibits the expression of HMGB3 in triple-negative breast cancer cells, significantly inhibits the proliferation, cloning ability, invasion and migration ability of triple-negative breast cancer cells, and promotes the apoptosis of triple-negative breast cancer cells. Therefore, coptisine hydrochloride can be used to prepare a drug for preventing and / or treating triple-negative breast cancer.

[0029] (2) The coptisine hydrochloride provided by the present invention can also enhance the drug sensitivity of cisplatin. The combined use of coptisine 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings, "*" represents P<0.05, and there is statistical significance between the groups:

[0031] Figure 1 Shows the expression of HMGB3 in triple-negative breast cancer tissues and triple-negative breast cancer cell lines. Figure 1 In the figure, A: GEPIA2 online database analyzed the expression level of HMGB3 in breast cancer tumor tissues and normal tissues; B: GEO dataset GSE45827 analyzed the expression level of HMGB3 in different types of breast cancer; C: GSE58812 analyzed the survival prognosis of patients with triple-negative breast cancer with high and low expression of HMGB3; D: Immunohistochemistry analysis of HMGB3 expression levels in different types of breast cancer; E: Western blot experiment detected the expression of HMGB3 in different types of breast cancer cell lines; F, G: Construction of HMGB3 knockdown and overexpression cell lines and verification by Western blot experiment.

[0032] Figure 2 It shows 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 migration and invasion of triple-negative breast cancer cells. Figure 3 In the figure, A: Western blot assay was used to detect the effect of HMGB3 on the migration and invasion of triple-negative breast cancer cells; B, C: Transwell assay was used to detect the effect of HMGB3 on the migration and invasion of triple-negative breast cancer cells.

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

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

[0036] Figure 6 It showed that coptisine hydrochloride could inhibit the protein expression level of HMGB3. Figure 6 In the study, Western blot assay was used to detect the expression of HMGB3 in triple-negative breast cancer cells after treatment with different concentrations of coptisine hydrochloride.

[0037] Figure 7 It was shown that coptisine hydrochloride can inhibit the proliferation, migration and invasion of triple-negative breast cancer cells and promote the apoptosis of triple-negative breast cancer cells. Figure 7 In the figure, A: CCK-8 method was used to detect the proliferation of triple-negative breast cancer cells after treatment with coptisine hydrochloride at different concentrations and for different time periods; B, C: coptisine hydrochloride can inhibit the invasion and formation ability of triple-negative breast cancer cells; D: CCK-8 method was used to detect the inhibitory ability of coptisine hydrochloride at different concentrations and combined with cisplatin on cells.

[0038] Figure 8 It was shown that coptisine hydrochloride can enhance the anti-tumor activity mediated by cisplatin. Figure 8 A: CCK-8 assay to detect the cell proliferation of HCC1937 and BT549 after cisplatin combined with coptisine hydrochloride; B: Transwell assay to detect the cell invasion after cisplatin combined with coptisine hydrochloride; C: Comet assay to verify the cell DNA damage after cisplatin combined with coptisine hydrochloride; D: Mouse tumor size at the end point of the transplanted tumor model experiment; E: Mouse tumor weight statistics; F: Mouse tumor volume change curve. DETAILED DESCRIPTION

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

[0040] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0042] Example 1

[0043] Pharmacodynamics experiments

[0044] 1. Experimental Methods

[0045] 1.1 Cell proliferation experiment: CCK-8 method: Triple-negative breast cancer cells were inoculated in 96-well plates, and cells were treated with coptisine hydrochloride at different concentrations (0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM). After culturing for 24, 48, and 72 hours, 10 μl of CCK-8 reagent was added to each well, incubated at 37 ° C for 2 hours, and the absorbance at 450 nm was measured to evaluate the proliferation ability of cells. EdU cell proliferation experiment: Triple-negative breast cancer cells were inoculated in 96-well plates, and EdU fluorescent markers were added to the cell culture medium after the cells adhered to the wall, incubated at 37 ° C for 4 hours, and fixed with 4% paraformaldehyde for 20 minutes. The fixed cells were permeabilized for 10 minutes to allow the fluorescent marker to penetrate the cell membrane. Use the EdU detection kit to perform the following operations according to the instructions. Finally, the fluorescence signal of the cells was observed using a fluorescence microscope to determine the uptake of EdU in the cells and then judge the proliferation activity of the cells.

[0046] 1.2 Transwell experiment: Prepare a 24-well plate, add 500 μl of complete culture medium to each well, and then put it into the Transwell chamber. The chamber for the invasion experiment contains matrix gel, and the chamber for the migration experiment does not contain matrix gel. According to the migration and invasion characteristics of different cells, serum-free culture medium is used to culture at 37°C for several hours to several days. After the culture is completed, the chamber is removed, the culture medium is discarded, washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, and stained with crystal violet for 20 minutes. After the staining is completed, the upper cells of the chamber are scraped off with a cotton swab, cleaned, dried at room temperature, photographed, and counted. The migration and invasion ability of cells is evaluated by analyzing the number and morphological characteristics of migrating or invading cells.

[0047] 1.3 Nude mouse tumor bearing experiment: The Balb / c nude mice used in this experiment were 4-6 weeks old female mice. HCC1937 triple negative breast cancer cells (5×10 6100 μl suspension was made from 100 μl of ...

[0048] 1.4 Western blot: Collect triple-negative breast cancer cells, use RIPA lysis buffer and add protease inhibitors and phosphatase inhibitors, centrifuge at 4°C 12000rpm for 30 minutes to obtain protein supernatant, and detect protein concentration by BCA method. The protein sample is separated and transferred by SDS-PAGE electrophoresis, and then the PVDF membrane is blocked and incubated with antibodies. Finally, by adding a colorimetric substrate, the expression of the protein is observed under optical conditions. Through the above steps, the expression level of the target protein in the sample can be detected, and semi-quantitative or quantitative analysis can be performed.

[0049] 1.5 Comet Assay: Collect cells, remove the culture medium after centrifugation, and resuspend the cells in PBS. Dilute the cells to 1×10 6 cells / mL. Mix the cell suspension with low melting point agarose and quickly drop the mixture onto a pre-cooled slide to form a thin layer and wait for it to solidify. Place the slide in an electrophoresis tank, pour in the electrophoresis buffer, and apply a voltage of 25V for 30 minutes. Stain with fluorescent dye to visualize DNA. Wash with PBS after staining to remove unbound dye. Observe the slide using a fluorescent 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 the expression of HMGB3 in different types of breast cancer. Tissue samples were fixed on slides and dehydrated and dewaxed to maintain the structural integrity of the samples. Antibodies were used to detect specific antigenic proteins. The target protein was labeled by adding a dye to make it visible under a microscope. The stained samples were observed under a microscope to determine the presence and distribution of the target protein. Immunohistochemistry staining was scored according to the following criteria: "-" indicates no positively stained cells, "+" indicates 1-20% positive cells, "++" indicates 20-50% positive cells, and "+++" indicates 50-100% positive cells. IHC expression of the protein was evaluated: "-~+" was defined as low expression; "++~+++" was defined as high expression.

[0051] 2. Experimental results

[0052] 2.1 Expression of HMGB3 in triple-negative breast cancer tissues and triple-negative breast cancer cell lines. Figure 1 As shown in Figure A, in order to analyze the expression of HMGB3 in breast cancer and normal tissues, the GEPIA2 online database was used for analysis. Compared with normal breast cancer tissues, the mRNA expression level of HMGB3 in breast cancer tissues was significantly increased (P<0.05). Figure 1 As shown in Figure B, in order to explore the expression of HMGB3 in different types of breast cancer, the GEO public dataset (GSE45827) was used for bioinformatics analysis and it was found that triple-negative breast cancer was accompanied by a higher level of HMGB3 expression. Figure 1 As shown in Figure C, in order to analyze the relationship between HMGB3 and the prognosis of patients with triple-negative breast cancer, the GEO public dataset (GSE58812) was used for bioinformatics analysis and it was found that patients with triple-negative breast cancer who highly expressed HMGB3 had a poor prognosis.

[0053] In order to further verify the difference in the expression of HMGB3 in different types of breast cancer, Figure 1 As shown in Figure D, the expression of HMGB3 in breast cancer tissues of different types was analyzed by immunohistochemistry, and the expression level of HMGB3 in triple-negative breast cancer tissues was higher. Figure 1 As shown in Figure E, Western blot experiments revealed that among breast cancer cell lines of different types, the protein expression level of HMGB3 in triple-negative breast cancer cell lines HCC1937 and BT549 was significantly higher. Figure 1 As shown in F and G, HMGB3 knockdown and overexpression (HCC1937 and BT549 cells) cell lines were constructed using lentivirus, and the above HMGB3 expression results were verified by Western blot experiments.

[0054] 2.2HMGB3 promotes the proliferation of triple-negative breast cancer cells and inhibits the apoptosis of triple-negative breast cancer cells. In order to explore whether HMGB3 can affect the proliferation ability of triple-negative breast cancer cells, EdU experiments were performed to detect cell proliferation ability. Figure 2 As shown in Figure 4A, EdU experiments showed that 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 shown in Figure E, the Balb / c Nude mice used in the experiment were 4-6 weeks old female mice. The constructed HCC1937 (Ctrl, Sh-HMGB3#1, Sh-HMGB3#2) triple-negative breast cancer cells (5×10 6100 μl suspension was made from 100 μl of ... Figure 2 As shown in F and G, knocking down HMGB3 expression significantly inhibited the growth of subcutaneous transplanted tumors in mice.

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

[0056] 2.4HMGB3 can reduce cisplatin-mediated anti-tumor activity. Next, in order to further explore the role of HMGB3 in triple-negative breast cancer, triple-negative breast cancer BT549 cells were selected to construct an HMGB3 knockdown cell line, 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 usually show good sensitivity in the early stage of cisplatin treatment, which is mainly attributed to their defective DNA damage repair mechanism. Patients with advanced triple-negative breast cancer often show cisplatin resistance. Figure 4 As shown in B, CCK-8 experiments demonstrated that cisplatin promoted the apoptosis of triple-negative breast cancer cells HCC1937 and BT549, and the inhibitory effect increased with increasing concentration.

[0057] 2.5HMGB3 attenuates cisplatin-mediated anti-triple-negative breast cancer activity. Figure 5 It can be seen that the comet assay found that compared with the control group, the DNA damage of triple-negative breast cancer cells treated with cisplatin increased significantly after knocking down HMGB3. The above suggests that knocking out HMGB3 in vivo can increase the sensitivity of tumor cells to cisplatin treatment.

[0058] 2.6 Coptis chinensis hydrochloride inhibits the expression of HMGB3. After HCC1937 and BT549 triple-negative breast cancer cells were treated with different concentrations of coptis chinensis hydrochloride (0 μM, 10 μM, 20 μM, 40 μM, 80 μM) for 24 hours, the cells were collected to extract proteins, and the expression of HMGB3 was detected by Western blot. Figure 6 It can be seen that coptisine hydrochloride can inhibit the expression of HMGB3 in triple-negative breast cancer cells in a dose-dependent manner. The higher the drug concentration, the stronger the effect of inhibiting HMGB3 expression.

[0059] 2.7 Coptis chinensis hydrochloride inhibits the proliferation, migration and invasion of triple-negative breast cancer cells and promotes the apoptosis of triple-negative breast cancer cells. HCC1937 and BT549 cells were treated with coptis chinensis hydrochloride at different concentrations (0 μM, 10 μM, and 20 μM). CCK-8 was used to detect the growth and proliferation of HCC1937 and BT549 cells. Figure 7 As shown in Figure A, coptisine hydrochloride significantly inhibited the survival rate of HCC1937 and BT549 cells in a dose-concentration- and time-dependent manner. Transwell assay was used to detect the migration and invasion abilities of HCC1937 and BT549 cells. Figure 7 As shown in Figures B and C, coptisine hydrochloride inhibited the migration and invasion of HCC1937 and BT549 cells, and the inhibitory effect increased with increasing concentration. Figure 7 As shown in D, the CCK-8 experiment proved that low-dose coptisine hydrochloride combined with cisplatin significantly inhibited cell growth in triple-negative breast cancer.

[0060] 2.8 Coptis chinensis hydrochloride enhances the therapeutic sensitivity of drug-resistant cell lines to cisplatin. In order to verify that coptis chinensis hydrochloride enhances the therapeutic sensitivity of triple-negative breast cancer cell lines HCC1937 and BT549 to cisplatin. Experimental group 1 was added with different concentrations of cisplatin (5μM, 10μM, 20μM, 30μM); experimental group 2 was added with different concentrations of coptis chinensis hydrochloride (5μM, 10μM, 15μM, 20μM); experimental group 3 was added with a fixed concentration (5μM) of coptis chinensis hydrochloride and different concentrations of cisplatin (5μM, 10μM, 20μM, 30μM); culture medium was used as a blank group; 0.1% DMSO culture medium was used as a control group. After 72 hours of action, 10μl of CCK-8 reagent was added to each well, incubated at 37°C for 2 hours, and the 96-well plate was taken out and the absorbance value at 450nm was detected using an enzyme 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. CompuSyn software was used to calculate the combination index CI, and CI ≤ 0.9 was considered to have a potential synergistic effect between the two drugs. The results are shown in Table 1.

[0061] Table 1: Determination results of the proliferation inhibition rate of triple-negative breast cancer cells using cisplatin, coptisine hydrochloride and their combination

[0062]

[0063] It can be seen from Table 1 that when coptisine hydrochloride is used alone, the inhibition rate of triple-negative breast cancer cell proliferation increases with the increase of drug concentration. And the inhibition rate of triple-negative breast cancer cell proliferation in combination with coptisine hydrochloride and cisplatin is significantly higher than that in the single-drug group, which shows that the combination of coptisine hydrochloride and cisplatin has a strong synergistic effect in inhibiting the proliferation of triple-negative breast cancer cells.

[0064] Figure 8 A in the middle is the survival rate of tumor cells detected by using the drug concentrations in Table 1. The CCK-8 method confirmed that after the combination of cisplatin and coptisine hydrochloride, the sensitivity of the drug-resistant cell lines HCC1937 and BT549 to cisplatin was enhanced. The migration and invasion abilities of HCC1937 and BT549 cells were detected by Transwell assay. Figure 8 As shown in Figure B, coptisine hydrochloride combined with cisplatin inhibited the invasion ability of HCC1937 and BT549 cells. Figure 8 As shown in C, comet assay confirmed that coptisine hydrochloride combined with cisplatin significantly increased DNA damage in triple-negative breast cancer cell lines HCC1937 and BT549.

[0065] Next, the inventors used nude mice in vivo tumor bearing experiments to evaluate the anti-tumor effect of coptisine hydrochloride combined with cisplatin. The Balb / c nude mice used were 4-6 weeks old female mice. HCC1937 triple negative breast cancer cells (5×10 6 Cells) were made into a 100μl suspension and injected into the back of nude mice. One week after the tumor was formed, the mice were randomly divided into 4 groups, including the control group, the intraperitoneal injection of cisplatin group (25mg / kg), the intraperitoneal injection of coptisine hydrochloride group (8mg / kg), and the cisplatin and coptisine hydrochloride combination group (cisplatin 25mg / kg combined with coptisine hydrochloride 8mg / kg). The drugs were administered continuously for 3 weeks. The weight of mice and the size of tumors were measured regularly. The volume of the tumor was calculated as: length × width × width × 0.5. After 3 weeks of administration, the nude mice were killed, the tumors were peeled off, weighed, and photographed. Figure 8 As shown in Figure DF, the combination of coptisine hydrochloride and cisplatin can significantly inhibit the tumor volume.

[0066] In summary, the present invention found that coptisine hydrochloride significantly inhibited the expression level of HMGB3 in triple-negative breast cancer cells, significantly inhibited the proliferation, cloning ability, invasion and migration ability of triple-negative breast cancer cells, and promoted the apoptosis of triple-negative breast cancer cells. Secondly, coptisine hydrochloride can also enhance the drug sensitivity of cisplatin. The combination of coptisine 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.

[0067] Obviously, the above embodiments are only examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. Those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, 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 is also intended to include these modifications and variations.

Claims

1. Use of coptisine hydrochloride in the preparation of a drug for preventing and / or treating triple-negative breast cancer, characterized in that: The molecular formula of the coptisine hydrochloride is C 19 H 14 ClNO4, the chemical structure is as follows:

2. The use according to claim 1, characterized in that The coptisine hydrochloride prevents and / or treats triple-negative breast cancer by inhibiting the expression of HMGB3.

3. The use according to claim 1, characterized in that The coptisine hydrochloride prevents and / or treats triple-negative breast cancer by inhibiting the proliferation, invasion and migration capabilities of triple-negative breast cancer cells and promoting the apoptosis of triple-negative breast cancer cells.

4. Use of coptisine hydrochloride in the preparation of HMGB3 inhibitors.

5. Use of coptisine hydrochloride in the preparation of drugs for inhibiting or knocking out HMGB3.

6. A pharmaceutical composition for preventing and / or treating triple-negative breast cancer, characterized in that: The pharmaceutical composition comprises cisplatin and coptisine hydrochloride, and the molar ratio of the cisplatin to the coptisine hydrochloride is 1-4:

1.

7. The pharmaceutical composition according to claim 6, characterized in that The molar ratio of the cisplatin to the coptisine hydrochloride is 1.5-4:

1.

8. Use of the pharmaceutical composition according to claim 6 or claim 7 in the preparation of a medicament for preventing and / or treating triple-negative breast cancer.

9. The use according to claim 8, characterized in that The cisplatin and coptisine hydrochloride in the pharmaceutical composition are prepared into an oral preparation with a pharmaceutically acceptable carrier.

10. The use according to claim 9, characterized in that The oral preparation is a tablet, a capsule, a granule or an oral liquid.

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