Application of traditional Chinese medicine monomer syrian a in preparation of anti-prostate cancer drugs

Abstract

The application discloses application of a traditional Chinese medicine monomer, syringa pubescens A, in preparation of a medicine for resisting prostate cancer. The application proves through experiments that syringa pubescens A can inhibit the proliferation, migration and invasion of DU145 cells and promote cell apoptosis by down-regulating the expression of Atox1 protein and activating the activity of a copper death signal pathway. Based on the above discovery, syringa pubescens A has an anti-tumor effect on DU145 cells and can be used as a potential candidate medicine for resisting prostate cancer, thereby providing a new medicine selection for the treatment of prostate cancer.

Description

Technical Field

[0001] This invention belongs to the field of prostate cancer treatment drug technology, and specifically relates to the application of the traditional Chinese medicine monomer Astragalus complanatus glycoside A in the preparation of anti-prostate cancer drugs. Background Technology

[0002] Most newly diagnosed prostate cancer patients have localized prostate cancer and receive androgen deprivation therapy (ADT) followed by radical prostatectomy or radiation therapy. However, due to AR amplification, mutations, splicing variations, and the emergence of compensatory pathways, some patients still experience disease recurrence or progression after treatment. Studies show that most patients experience progression from androgen-dependent prostate cancer to metastatic castration-resistant prostate cancer. Although patients with metastatic castration-resistant prostate cancer currently benefit from a wide range of effective treatment options, metastatic castration-resistant prostate cancer remains incurable, with a very poor prognosis and a 5-year survival rate of only 30%. Therefore, metastatic castration-resistant prostate cancer remains an incurable disease and a cause of high mortality, urgently requiring the search for new therapeutic agents. Summary of the Invention

[0003] In view of the shortcomings of existing technologies, this invention provides the application of the traditional Chinese medicine monomer Astragalus complanatus glycoside A in the preparation of anti-prostate cancer drugs. This invention discovers that Astragalus complanatus glycoside A can inhibit the expression of ATOX1 protein, promote copper accumulation and copper death in cells, inhibit the proliferation, invasion, and damage repair of DU145 cells, and simultaneously promote apoptosis. As a traditional Chinese medicine monomer, it has the advantages of low toxicity and few side effects.

[0004] This invention provides the use of Astragalus complanatus glycoside A in the preparation of a medicament for the prevention and / or treatment of prostate cancer.

[0005] According to a specific embodiment of the present invention, the drug is a drug that inhibits the proliferation, invasion, or migration of prostate cancer cells.

[0006] According to a specific embodiment of the present invention, the drug is a drug that promotes apoptosis of prostate cancer cells.

[0007] According to a specific embodiment of the present invention, the drug is a drug that reduces the expression level of Atox1 protein.

[0008] According to a specific embodiment of the present invention, the drug is a drug that increases the expression level of copper death-related protein.

[0009] According to a specific embodiment of the present invention, the copper death-related protein is DLAT protein and / or HSP70 protein.

[0010] According to a specific embodiment of the present invention, the drug is a formulation prepared with an effective amount of Astragaloside A as the main or sole active ingredient and pharmaceutically acceptable excipients.

[0011] According to a specific embodiment of the present invention, the formulation is any pharmaceutically acceptable dosage form.

[0012] According to a specific embodiment of the present invention, the preparation is an injection, and preferably, the concentration of Astragaloside A in the injection is 0.05-1.6 mM.

[0013] According to a specific embodiment of the present invention, the concentration of Astragalus complanatus glycoside A in the injection is 0.2–0.8 mM.

[0014] The beneficial effects of this invention are as follows:

[0015] This invention experimentally demonstrates that Astragalus complanatus glycoside A inhibits the proliferation, migration, and invasion of DU145 cells and promotes apoptosis by downregulating Atox1 protein expression and activating the copper death signaling pathway. Based on these findings, Astragalus complanatus glycoside A exhibits antitumor activity against DU145 cells and can serve as a potential candidate drug for prostate cancer treatment, providing a new drug option for prostate cancer therapy. Attached Figure Description

[0016] Figure 1 The results of experiments on the inhibition of prostate cancer cell proliferation by Astragalus complanatus glycoside A are presented. A. Chemical structural formula of Astragalus complanatus glycoside A; B. Effects of different concentrations and time intervals of Astragalus complanatus glycoside A solution on the proliferation ability of human prostate cancer cells DU145. * P<0.05, ** P<0.01; C. Cell images of human prostate cancer cells DU145 after treatment with different concentrations of Complanatoside A solution for 48 h, scale bar is 10 μm; where, Cell viability of DU145 (100% of control): cell proliferation rate relative to the control group (100%); Complanatoside A (mM): Complanatoside A (mmol / L); DMSO: control (dimethyl sulfoxide);

[0017] Figure 2 The results of the experiment on the inhibitory effect of complanatoside A on DU145 cell invasion were presented. A. Transwell assay was used to evaluate the effect of complanatoside A solution on DU145 cell invasion. The scale bar is 10 μm. Complanatoside A (mM): Complanatoside A (mmol / L); B. Number of invading cells. * P<0.05, ** P<0.01;

[0018] Figure 3The results of the experiment on the inhibitory effect of Astragalus complanatus A on the migration of DU145 cells are as follows: A. Scratch repair assay to evaluate the effect of Astragalus complanatus A solution on the migration of DU145 cells, scale bar is 10 μm; B. Scratch damage repair rate. * P<0.05, ** P<0.01. Wherein, Scratch healing ratio (%): scratch damage repair rate; Complanatoside A (mM): Complanatoside A (mmol / L);

[0019] Figure 4 To assess the effects of different concentrations of Astragalus complanatus A on DU145 cell apoptosis, the following methods were used: A. Flow cytometry was used to evaluate the effect of different concentrations of Astragalus complanatus A solution on DU145 cell apoptosis after 48 hours of treatment; B. Cell apoptosis rate. * P<0.05, ** P<0.01. Wherein, Apoptosis ratio (%): cell apoptosis rate; Complanatoside A (mM): Complanatoside A (mmol / L);

[0020] Figure 5 The results of the experiment on the inhibition of Atox1 protein expression by Astragalus complanatus glycoside A were as follows: A. Immunofluorescence assay to evaluate the effect of different concentrations of Astragalus complanatus glycoside A solution on Atox1 protein localization in DU145 cells after 48 h of treatment (scale bar: 10 μm); B. Average fluorescence intensity. * P<0.05, ** P<0.01. Mean fluorescence intensity; Complanatoside A (mM); C. Immunoblotting assessment of the effect of different concentrations of Complanatoside A solution on Atox1 protein expression in DU145 cells after 48 h of treatment;

[0021] Figure 6 To illustrate the results of experiments on the promotion of copper apoptosis in DU145 cells by Astragalus complanatus A, A. The effect of different concentrations of Astragalus complanatus A solution on the copper ion content in DU145 cells after 48 h of treatment. * P<0.05, ** P<0.01; B. Effect of different concentrations of scutellarin A solution on HSP70 protein expression in DU145 cells after 48 h of treatment; C. Effect of different concentrations of scutellarin A solution on DLAT protein expression in DU145 cells after 48 h of treatment;

[0022] Figure 7 The results of an in vivo experiment showing the inhibition of RM-1 cell growth by Astragalus complanatus glycoside A are shown in Figure A. A representative tumor image at the end of Astragalus complanatus glycoside A treatment; Figure B. Histogram of tumor weight changes.* P<0.05, ** P<0.01, *** P < 0.001, where: Tumor weight; C. Bar chart of tumor inhibition rate; D. Tumor growth curve. * P<0.05, ** P<0.01, *** P < 0.001, where Tumor volume is the volume of the tumor; E is the daily variation in total mouse weight. Mouse bodyweight is the body weight of the mouse. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Experimental materials and reagents: Human prostate cancer DU145 cells and mouse prostate cancer cells RM-1 (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.), cultured in 1640 medium supplemented with 10% fetal bovine serum, 5% penicillin and streptomycin (all purchased from Wuhan Pronosai Life Science Technology Co., Ltd.); 0.25% trypsin-EDTA digestion solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.); Astragalus complanatus glycoside A (purchased from Shanghai Yuanye Biotechnology Co., Ltd.); Atox1, DLAT, HSP70, GAPDH, FITC-labeled goat anti-rabbit IgG and other antibodies (purchased from Wuhan Sanying Biotechnology Co., Ltd.) The following reagents were purchased from Beijing Solarbio Science & Technology Co., Ltd.: BCA protein concentration assay kit, MTT assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.); BCA protein concentration assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.), ultrasensitive ECL chemiluminescence reagent kit (both purchased from Beyotime Biotechnology Co., Ltd.); Transwell-24 membrane nesting, Matrigel (base gel), 4% paraformaldehyde, 0.1% crystal violet (purchased from Beijing Solarbio Science & Technology Co., Ltd.); apoptosis detection kit (purchased from Beyotime Biotechnology (China) Co., Ltd.); poly-L-lysine, RIPA lysis buffer, ECL Plus ultrasensitive luminescence solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.); Elesclomol (ES), CuCl2 (Maclean Biotechnology Co., Ltd.).

[0025] Experimental equipment: CO2 cell incubator (Anhui Zhongke Duling Commercial Electric Appliance Co., Ltd.), ultra-clean workbench (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), high-speed refrigerated centrifuge (Eppendorf China), fluorescence microscope (Leica Microsystems (Shanghai) Trading Co., Ltd.), ELISA reader (Xiamen Jianfa High-Tech Co., Ltd.), flow cytometer (BD, USA).

[0026] Data analysis: All experiments were repeated at least three times. Data are expressed as mean ± standard deviation. SPSS 18.0 was used for analysis. The t-test was used to compare differences between groups, and P < 0.05 was considered statistically significant.

[0027] Example 1: Astragalus complanatus glycoside A inhibits the proliferation of DU145 cells.

[0028] The structural formula of Astragalus complanatus glycoside A is as follows: Figure 1 As shown in Figure A. The effect of different concentrations of Astragaloside A on the proliferation ability of prostate cancer cells was detected by the MTT assay. The specific steps are as follows: DU145 cells adherent to the wall were digested with 0.25% trypsin-EDTA digestion solution, and cultured at a concentration of 5 × 10⁻⁶ cells / cells. 3 Cells were seeded at a density of [number] cells / well in 96-well plates, with three replicates per group. After 24 hours, once the cells had fully adhered, 0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mmol / L of Astragalus complanatus A solution were added to the cell culture medium. After culturing for 24 hours and 48 hours, 90 μL of fresh culture medium was added, followed by 10 μL of MTT solution, and the cells were cultured for another 4 hours. The supernatant was then aspirated, and 100 μL of DMSO was added to each well. The plates were shaken at low speed for 10 minutes to dissolve any crystals. The absorbance at 490 nm was then measured using a microplate reader.

[0029] MTT assay results showed that astragaloside A inhibited the proliferation of DU145 cells in a concentration- and time-dependent manner, with a statistically significant difference compared to the control group (P<0.05). Its IC50 value was approximately 1.2 mM at 24 h and approximately 0.7 mM at 48 h. Figure 1 As shown in B.

[0030] Cellular changes were observed using microscopic photography. The specific steps were as follows: DU145 cells adhering to the culture vessel were prepared by enzymatic digestion with 0.25% trypsin-EDTA digestion solution, and cultured at a density of 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of [number] cells / well in 6-well plates, with 3 replicates per group. After 24 hours, once the cells had fully adhered, 0, 0.2, 0.4, and 0.8 mmol / L of Astragalus complanatus A solution were added to the cell culture medium, respectively. Cells were cultured for another 48 hours before microscopic observation and photography. Figure 1 As shown in C. The cell photographs show that the cells transitioned from an adherent to a non-adherent state and floated in the culture medium.

[0031] Example 2: Astragalus complanatus glycoside A inhibits DU145 cell invasion.

[0032] The effect of different concentrations of Astragalus complanatus glycoside A solution on the invasion of DU145 cells was detected using a Transwell assay. The specific procedure was as follows: First, Matrigel was diluted with serum-free medium at a ratio of 1:5, then added dropwise to the upper surface of a Transwell chamber and incubated at room temperature for 1 hour to coat the Transwell. DU145 cells were treated with Astragalus complanatus glycoside A solutions of 0, 0.2, 0.4, and 0.8 mmol / L for 48 hours. Cells were then digested with 0.25% trypsin-EDTA solution to prepare a cell suspension. After centrifugation, the medium was removed, and cell culture medium containing 1% FBS was added to adjust the cell density to 5 × 10⁶ cells / mL. 6 Cells / mL. 100 μL of cell suspension was added to the upper chamber of the Transwell after treatment with Matrigel, and 600 μL of cell culture medium containing 10% FBS was added to the lower chamber. After 48 h, the chamber was removed, fixed with 4% paraformaldehyde for 30 min, washed with water, and the cells on the upper chamber were removed with a cotton swab. Then, the cells were stained with 0.1% crystal violet for 20 min, air-dried, and photographed and counted in three fields of view under an inverted microscope.

[0033] The results showed that after treating DU145 cells with 0, 0.2, 0.4, and 0.8 mmol / L Astragalus complanatus A solutions for 48 h, Astragalus complanatus A inhibited cell invasion in a concentration-dependent manner, with significant differences compared to the control group. * P<0.05). For example... Figure 2 As shown in A and 2B.

[0034] Example 3: Astragalus complanatus glycoside A inhibits DU145 cell migration.

[0035] The effect of different concentrations of Astragalus complanatus glycoside A solution on the migration of DU145 cells was detected using a scratch repair assay. The specific procedure was as follows: adherent DU145 cells were prepared by enzymatic digestion with 0.25% trypsin-EDTA digestion solution, and the cells were then cultured at a concentration of 5 × 10⁶ cells / year. 4 DU145 cells were seeded at a density of 100 cells / well in 6-well plates, with 3 replicates per group. After 24 hours, once the cells had fully adhered to the plates, a straight line was drawn in the center of each well using a 200 μL pipette tip. The culture medium was then replaced, and the cells were observed and photographed under a microscope. DU145 cells were then treated with 0, 0.2, 0.4, and 0.8 mmol / L Astragalus complanatus A solution for 48 hours, and the cells were observed and photographed under a microscope afterward.

[0036] The results showed that after treating DU145 cells with 0, 0.2, 0.4, and 0.8 mmol / L astragaloside A solutions for 48 h, astragaloside A inhibited cell migration in a concentration-dependent manner, with significant differences compared to the control group.* P<0.05). For example... Figure 3 As shown in A and 3B.

[0037] Example 4: Astragalus glycoside A promotes apoptosis in DU145 cells.

[0038] The effect of different concentrations of Astragalus complanatus glycoside A solution on apoptosis of DU145 cells was detected by flow cytometry. The specific steps are as follows: DU145 cells adherent to the culture vessel were digested with 0.25% trypsin-EDTA digestion solution to prepare a cell suspension, and the suspension was then diluted at 5 × 10⁻⁶ cells / mL. 4 DU145 cells were seeded into 6-well plates at a density of 10 cells / well, with 3 replicates per group. After 24 hours, once the cells had fully adhered, they were treated with 0, 0.2, 0.4, and 0.8 mmol / L Astragalus complanatus A solution for 48 hours. The adhered DU145 cells were then digested with 0.25% trypsin-EDTA solution to prepare a cell suspension. The suspended cells were collected by centrifugation at approximately 300×g, 2-8℃, for 5 minutes, and the culture medium was discarded. The cells were washed twice with cold PBS. The cells were resuspended in 400 μL of 1X Annexin V binding buffer to a concentration of approximately 1×10⁻⁶. 6 cells / ml. Add 5 μl of Annexin V-FITC staining solution to the cell suspension, mix gently, and incubate at 2-8°C in the dark for 15 minutes. Add 5-10 μl of PI staining solution, mix gently, and incubate at 2-8°C in the dark for 2-5 minutes. Analyze immediately using flow cytometry.

[0039] The results showed that after treating DU145 cells with 0, 0.2, 0.4, and 0.8 mmol / L astragaloside A solutions for 48 h, astragaloside A promoted cell apoptosis in a concentration-dependent manner, with significant differences compared to the control group. * P<0.05). For example... Figure 4 As shown in A and 4B.

[0040] Example 5: Astragalus complanatin A inhibits Atox1 expression in DU145 cells.

[0041] The effects of different concentrations of Astragaloside A solution on the copper-stabilizing protein Atox1 in DU145 cells were detected using immunofluorescence and immunoblotting. The specific steps are as follows:

[0042] ① Immunofluorescence assay: After washing the coverslips with PBS and anhydrous ethanol, place them in a 6-well plate. Apply 0.01% poly-L-lysine to the coverslips to increase cell adhesion. After air drying, irradiate with UV light for 1 hour. DU145 cells were cultured at 2.0 × 10⁻⁶ cells / well. 5Cells were seeded at a density of [number] cells / mL in six-well plates. After cell adhesion, they were treated with 0, 0.2, 0.4, and 0.8 mmol / L Astragaloside A solution for 48 h. After the treatment time, the culture medium was discarded, and the cells were gently washed twice with PBS. Cells were fixed with 4% paraformaldehyde for 15 min, washed twice with PBS, and then incubated with 0.5% Triton X-100 at room temperature for 20 min to allow cell permeability. Cells were washed twice with PBS and then blocked with 0.5% Triton X-100 and 5% BSA in a humidified room temperature environment for 30 min. After blocking, Atox1 antibody was prepared at a ratio of 1:200 and incubated overnight at 4°C. Cells were washed twice with PBS and then incubated with FITC-labeled goat anti-rabbit IgG (1:200) antibody at 37°C in the dark for 1 h. Images were acquired using a fluorescence inverted microscope and analyzed using ImageJ.

[0043] ② Immunoblotting assay: DU145 cells were seeded at a depth of 25 cm. 2 In cell culture flasks, when the cell density reached 80%, 0, 0.2, 0.4, and 0.8 mmol / L of Astragalus complanatus A solution were added. After 48 hours, total protein from the drug-treated cells was extracted using a mixed lysis buffer of RIPA and PMSF, and protein quantification was performed using the BCA standard method. A 12% SDS-PAGE gel was prepared for electrophoresis, then transferred to a PVDF membrane. After blocking with 5% BSA for 2 hours, primary antibodies (Atox1 diluted 1:1000, GAPDH diluted 1:5000) were added and incubated overnight at 4°C in the dark. After washing three times with TBST, secondary antibody (anti-mouse IgG) was added and incubated for 1 hour. After washing three times with TBST, ECL chemiluminescence buffer was added, and the bands were exposed in an imaging system.

[0044] Immunofluorescence results showed that after treating DU145 cells with 0, 0.2, 0.4, and 0.8 mmol / L Astragaloside A solutions for 48 h, the intensity of green fluorescence was significantly reduced, showing a significant difference compared to the control group. * (P<0.05). The protein's localization remained unchanged. For example... Figure 5 As shown in A and 5B.

[0045] Immunoblotting results showed that after treating DU145 cells with 0, 0.2, 0.4, and 0.8 mmol / L Astragaloside A solutions for 48 h, the protein expression level of Atox1 decreased in a concentration-dependent manner, with significant differences compared to the control group. * P<0.05). For example... Figure 5 As shown in C.

[0046] Example 6: Astragalus glycoside A promotes copper death in DU145 cells

[0047] The effects of different concentrations of Astragalus complanatus glycoside A solution on copper content and copper death in DU145 cells were detected by copper ion content determination and immunoblotting. The specific steps are as follows:

[0048] ① Copper ion content determination: DU145 cells adherent to the culture vessel were digested with 0.25% trypsin-EDTA digestion solution to prepare a cell suspension, and the suspension was diluted with 5 × 10⁻⁶ cells. 4 DU145 cells were seeded into 6-well plates at a density of 10 cells / well, with 3 replicates per group. After 24 hours, once the cells had fully adhered, they were treated with 0, 0.2, 0.4, and 0.8 mmol / L Astragaloside A solution for 48 hours. The cell copper content was then determined according to the instructions of the cell copper (Cu) content assay kit.

[0049] ② Immunoblotting assay: DU145 cells were seeded at a depth of 25 cm. 2 In cell culture flasks, when the cell density reached 80%, 0, 0.2, 0.4, and 0.8 mmol / L of Astragalus complanatus A solution were added. After 48 hours, total protein from the drug-treated cells was extracted using a mixed lysis buffer of RIPA and PMSF, and protein quantification was performed using the BCA standard method. A 12% SDS-PAGE gel was prepared for electrophoresis, then transferred to a PVDF membrane. After blocking with 5% BSA for 2 hours, primary antibodies (DLAT diluted 1:1000, HSP70 diluted 1:1000, and GAPDH diluted 1:5000) were added and incubated overnight at 4°C in the dark. After washing three times with TBST, secondary antibody (anti-mouse IgG) was added and incubated for 1 hour. After washing three times with TBST, ECL chemiluminescence buffer was added, and the bands were exposed in an imaging system.

[0050] The results of copper ion content determination showed that after treating DU145 cells with 0, 0.2, 0.4, and 0.8 mmol / L Astragalus complanatus A solution for 48 h, the intracellular copper ion concentration increased in a concentration-dependent manner, with significant differences compared to the control group. * P<0.05). For example... Figure 6 As shown in Figure A.

[0051] Immunoblotting results showed that after treating DU145 cells with 0, 0.2, 0.4, and 0.8 mmol / L Astragaloside A solution for 48 h, the protein expression level of HSP70 increased in a concentration-dependent manner, with significant differences compared to the control group. * P<0.05), such as Figure 6 As shown in Figure B, after treating DU145 cells with 0, 0.2, 0.4, and 0.8 mmol / L Astragaloside A solutions for 48 h, the protein expression level of DLAT increased in a concentration-dependent manner, showing a significant difference compared to the control group. * P<0.05), such as Figure 6 As shown in C.

[0052] Example 7: Astragalus complanatus glycoside A inhibits tumor growth in vivo.

[0053] First, a mouse tumor model of RM-1 cell transplantation was established: 24 male C57BL / 6 mice, each weighing approximately 20±2g and aged five weeks, were used; three vials (approximately 1ml each) of RM-1 cell suspension in the logarithmic growth phase were taken, and the cell density was adjusted to 1×10⁻⁶. 9 / ml; Hair was removed from the left axilla of mice beforehand. After hair removal, the hair removal cream was wiped off with a cotton ball, and the area was disinfected with 75% ethanol after drying. Under these conditions, 120 μL (approximately 1.2 × 10⁻⁶) was subcutaneously injected into the left axilla of the mice. 8 Mice were randomly divided into a blank control group, a high-dose group of Astragalus complanatus A (50 mg / kg), a low-dose group of Astragalus complanatus A (25 mg / kg), and an ES-CU group (10 mg / kg) after inoculation. Nine days after inoculation, the tumors grew to the size of a grain of rice visible to the naked eye. Thirty mice with good tumor growth were randomly selected and divided into five groups. The administration period was 20 days, with administration every two days via intraperitoneal injection. Tumor volume and body weight were measured every two days. After 20 days of administration, the tumor tissue was removed, weighed, and the experimental data were recorded and calculated.

[0054] The results showed that after 20 days of injection of low-dose (25 mg / kg / day), high-dose (50 mg / kg / day) scutellarin A, and ES-CU, tumor growth, tumor weight, and volume in mice were significantly inhibited, with significant differences compared to the blank control group. * (P<0.05); Among them, the tumor inhibition rate of the low-dose astragaloside A group was 29.8%, the tumor inhibition rate of the high-dose astragaloside A group was 41.6%, and the tumor inhibition rate of the positive control group ES-CU was 49.8%. The results showed that there was no significant difference in effect between the high-dose experimental group and the positive control group; the total weight of the mice did not change. Figure 7 As shown in A, 7B, 7C, 7D, and 7E.

[0055] In summary, the applicant investigated whether astragaloside A could inhibit the proliferation, invasion, and migration of DU145 cells in vitro, and promote DU145 cell apoptosis. First, DU145 cells were treated with different concentrations of astragaloside A solution for 24 h and 48 h, respectively. The effect of astragaloside A on cell proliferation was detected by MTT assay. The results showed that the half-maximal inhibitory concentration (IC50) after 24 h of treatment was approximately 1.2 mmol / L, and the IC50 after 24 h of treatment was approximately 0.7 mmol / L. Compared with the control group, astragaloside A inhibited DU145 cell proliferation in a concentration- and time-dependent manner. Second, DU145 cells were treated with different concentrations of astragaloside A solution for 48 h, and the effect of astragaloside A solution on DU145 cell invasion was detected by Transwell assay. The results showed that astragaloside A solution inhibited DU145 cell invasion in a concentration gradient manner. Furthermore, the scratch wound healing of DU145 cells after treatment with different concentrations of Astragalus complanatus A solution for 48 hours was examined using a cell scratch assay. The results showed that Astragalus complanatus A solution inhibited the migration of DU145 cells in a concentration gradient. Finally, DU145 cells were treated with different concentrations of Astragalus complanatus A solution for 48 hours, and changes in cell apoptosis were detected by flow cytometry. The results showed that Astragalus complanatus A solution promoted apoptosis of DU145 cells in a concentration gradient. These results indicate that Astragalus complanatus A has the functions of inhibiting the proliferation, invasion, and migration of DU145 cells and promoting cell apoptosis.

[0056] Atox1 plays a crucial role in tumor progression, but it remains unclear whether astragaloside A can influence prostate cancer progression by regulating Atox1 expression. This invention explores whether the inhibitory function of astragaloside A on DU145 cells is related to Atox1. First, DU145 cells were treated with different concentrations of astragaloside A solution for 48 hours. Then, immunofluorescence was used to detect the fluorescence intensity and subcellular localization of Atox1. The results showed that, compared with the control group, astragaloside A reduced the fluorescence intensity of Atox1 but had no effect on cell localization. Further immunoblotting was used to detect the protein expression level of Atox1. The results showed that, compared with the control group, astragaloside A reduced the protein content of Atox1 in a concentration-dependent manner.

[0057] Copper death is a newly discovered cell death pattern and a novel target for cancer therapy. However, it remains unknown whether astragaloside A can influence copper levels by regulating Atox1 expression, thereby promoting copper death. Copper content measurements showed that astragaloside A significantly increased intracellular copper ion levels in a concentration-dependent manner compared to the control group. Furthermore, immunoblotting results indicated a significant increase in the expression levels of copper death-related proteins DLAT and HSP70 compared to the control group. Therefore, these results suggest that astragaloside A can inhibit Atox1 expression, promote intracellular copper accumulation, and ultimately lead to copper death.

[0058] Prostate cancer is one of the most common malignant tumors of the male genitourinary system. Traditional androgen deprivation therapy (ADT) inhibits tumor cell growth by lowering androgen levels. However, ADT suffers from problems such as drug resistance, serious side effects (e.g., osteoporosis, cardiovascular disease), and decreased quality of life for patients. Metastatic castration-resistant prostate cancer remains incurable and a major cause of mortality, urgently requiring the search for new treatment methods. Traditional Chinese medicine (TCM) has unique advantages in effectively improving the weakness symptoms of prostate cancer patients, regulating immune function, controlling tumor cell apoptosis and inhibiting tumor angiogenesis, and significantly improving patients' quality of life. Compared to ADT, TCM has gradually attracted the attention and importance of researchers due to its multi-target and low toxicity characteristics. Astragalus glycoside A is an active ingredient extracted from the traditional Chinese medicine Astragalus complanatus. As a monomer of TCM, it has low toxicity and almost no side effects. It can not only inhibit the proliferation, invasion, and migration of prostate cancer cells and promote apoptosis of prostate cancer cells, but also promote the accumulation of intracellular copper by inhibiting Atox1 expression, thereby leading to copper death in cells. Therefore, it can be seen that Astragalus complanatus glycoside A can inhibit tumor cell growth in multiple ways, improve treatment efficacy, and reduce the probability of drug resistance. Furthermore, its unique mechanism of using copper ions to kill cancer cells provides a new pathway for killing prostate cancer cells.

[0059] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. Use of astragalin A in the preparation of a medicament for preventing and / or treating prostate cancer, wherein the medicament is a medicament for inhibiting proliferation, invasion or migration of prostate cancer cells, or a medicament for promoting apoptosis of prostate cancer cells.

2. Use according to claim 1, characterized in that, The medicament is a medicament for reducing the expression level of Atox1 protein.

3. Use according to claim 1, characterized in that, The medicament is a medicament for increasing the expression level of copper death related protein.

4. Use according to claim 3, characterized in that, The copper death related protein is DLAT protein and / or HSP70 protein.

5. The use according to claim 1, characterized in that, The medicament is a preparation prepared from an effective amount of astragalin A as the main or only active ingredient and pharmaceutically acceptable adjuvants.

6. Use according to claim 5, characterized in that, The preparation is any dosage form that is pharmaceutically acceptable.

7. Use according to claim 6, characterized in that, The preparation is an injection.

8. Use according to claim 7, characterized in that, In the injection, the concentration of astragalin A is 0.05-1.6 mM.

9. Use according to claim 8, characterized in that, In the injection, the concentration of astragalin A is 0.2-0.8 mM.

Images