Application of amentoflavone in preparation of medicine for treating bladder cancer

Abstract

The invention provides application of amentotaxus biflavone in preparation of a medicine for treating bladder cancer, and provides application of amentotaxus biflavone in preparation of a medicine for treating bladder cancer, which has the advantages that by increasing the concentration of amentotaxus biflavone, the number of bladder cancer cells is gradually reduced, the cells are shrunk, nuclear shrinkage of different degrees is caused, the activity, proliferation, invasion and migration of bladder cancer T24 cells and bladder cancer 5637 cells are inhibited, and the bladder cancer is treated. The PI3K / AKT / NF-kappa B pathway promotes the expression of a pro-apoptosis gene Bax in bladder cancer cells, inhibits the expression of an apoptosis inhibitor gene Bcl-2, inhibits the proliferation of the bladder cancer cells by inducing G1 phase retardation of the bladder cancer cells, induces and reduces the expression of PI3K, AKT and NF-kappa B proteins in cell nucleuses through the PI3K / AKT / NF-kappa B pathway, and induces the apoptosis of the bladder cancer cells. The invention belongs to the field of medicine.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically involving the application of paclitaxel flavonoids in drugs for the prevention and treatment of bladder cancer. Background Technology

[0002] Bladder cancer (BCa) is the most common malignant tumor of the urinary system and the 10th most common cancer type worldwide. For the treatment of bladder cancer, transurethral resection of bladder tumor (TURP) is the basic treatment for non-muscle-invasive bladder cancer, and intravesical instillation therapy can further improve tumor prognosis. For muscle-invasive bladder cancer, radical cystectomy with neoadjuvant chemotherapy is the standard treatment, and optimal treatment includes a multidisciplinary approach encompassing surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy. Despite the use of multidisciplinary treatment methods, approximately 50% of patients with muscle-invasive bladder cancer develop metastasis within 2 years. Therefore, in-depth research into the underlying molecular mechanisms of bladder cancer and novel strategies to improve patient survival is crucial.

[0003] In Asia, Traditional Chinese Medicine (TCM) has been widely accepted as a supplementary and alternative form of cancer treatment. It is one of the most popular adjuvant therapies after radical surgery for tumors in East Asia. With its significant advantages such as multiple components, multiple targets, natural ingredients, and fewer side effects, it is increasingly attracting attention worldwide. Both retrospective and prospective studies have confirmed the therapeutic effects of TCM on tumors. Currently, TCM is widely used in the treatment of various tumors, demonstrating significant therapeutic effects in both scientific research and clinical applications.

[0004] Taxus chinensis flavonoids possess anti-inflammatory, antioxidant, anti-diabetic, anti-aging, and antiviral activities, as well as effects on the central nervous system and cardiovascular system. However, their application in inhibiting the viability and proliferation of bladder cancer T24 cells and bladder cancer 5637 cells, and inducing apoptosis in bladder cancer cells, has not yet been observed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of paclitaxel flavonoids in the preparation of drugs for treating bladder cancer. This invention provides that by increasing the concentration of paclitaxel flavonoids, the number of bladder cancer cells gradually decreases, cells shrink, and nuclear pyknosis occurs to varying degrees, inhibiting the activity and proliferation of bladder cancer T24 cells and bladder cancer 5637 cells, and thus can be used to prepare drugs for treating bladder cancer, belonging to the pharmaceutical field.

[0006] The technical solution of this invention: Application of Taxodium biflavonoids in the preparation of drugs for treating bladder cancer.

[0007] The structural formula of the aforementioned paclitaxel flavonoid is:

[0008] The aforementioned cypermethrin flavonoids inhibit the activity, proliferation, invasion, and migration of bladder cancer cells.

[0009] The aforementioned cyperus flavonoids induce apoptosis in bladder cancer cells by promoting the expression of the pro-apoptotic gene Bax and inhibiting the expression of the apoptosis-inhibiting gene Bcl-2.

[0010] The aforementioned cypermethrin flavonoids inhibit the proliferation of bladder cancer cells by arresting them in the G1 phase and promote the expression of the cell cycle inhibitor p21.

[0011] The aforementioned cyperus flavonoids induce apoptosis in bladder cancer cells by reducing the expression of PI3K, AKT, and NF-κB proteins in the cell nucleus through the PI3K / AKT / NF-κB pathway.

[0012] The aforementioned bladder cancer cells are bladder cancer T24 cells and bladder cancer 5637 cells.

[0013] The aforementioned drug, Taxodium flavonoids, is used as the sole active ingredient and is incorporated into a pharmaceutically acceptable carrier and / or excipients to form a pharmaceutically acceptable dosage form.

[0014] The aforementioned dosage forms are tablets, capsules, granules, oral liquids, suspensions, or injections.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to propose that paclitaxel flavonoids inhibit the viability and proliferation of bladder cancer T24 and bladder cancer 5637 cells through specific cytotoxic effects on T24 and 5637 cells, which can be used to prepare drugs for the treatment of bladder cancer. The high-dose group of paclitaxel flavonoids reduced the proliferation rate of bladder cancer cells by 44% compared with the blank control group, and the apoptosis rate of bladder cancer cells reached more than 13%. At the same time, paclitaxel flavonoids inhibited the migration of bladder cancer cells 563 and T24. Attached Figure Description

[0016] Figure 1 Cell viability plots (A: Inhibition of 5637 cell viability by paclitaxel flavonoids; B: IC50 bar graph of paclitaxel flavonoids inhibiting 5637 cell viability; C: Inhibition of T24 cell viability by paclitaxel flavonoids; D: IC50 bar graph of paclitaxel flavonoids inhibiting 5637 cell viability). Figure 2 Morphological changes in bladder cancer cells after treatment with cytoplasmic flavonoids (×10); Figure 3 : Proliferation of bladder cancer cells after treatment with cytoplasmic flavonoids and EUD statistical analysis (A: EdU measurement of bladder cancer cell proliferation 24 hours after treatment with cytoplasmic flavonoids; B: EUD statistical analysis of 5637 cells; C: EUD statistical analysis of T24 cells). Figure 4 Flow cytometry and cell cycle statistics of bladder cancer cells (A: Flow cytometry of bladder cancer cells 24 hours after treatment with Taxodium styracifolium; B: Cell cycle statistics of 5637 cells; C: Cell cycle statistics of T24 cells). Figure 5 Flow cytometry and statistical analysis of apoptosis in bladder cancer cells (A: Flow cytometry of apoptosis-related bladder cancer cells after treatment with cypermethrin; B: Statistical analysis of apoptosis in 5637 cells; C: Statistical analysis of apoptosis in T24 cells). Figure 6 : Bladder cancer cell migration graphs and statistical analysis graphs (A: Bladder cancer cell migration 24 hours after treatment with Taxodium styracifolium; B: Statistical analysis graph of 5637 cell migration; C: Statistical analysis graph of T24 cell migration). Figure 7 Bladder cancer cell invasion graphs and statistical analysis graphs (A: Bladder cancer cell invasion 24 hours after treatment with Taxodium spp.; B: Statistical analysis graph of invasion of 5637 cells; C: Statistical analysis graph of invasion of T24 cells). Figure 8 Immunofluorescence localization and statistical analysis of bladder cancer cells (A: Immunofluorescence (10×) localization of PI3K; B: Statistical analysis of 5637 cells; C: Statistical analysis of T24 cells). Figure 9 : Expression and statistical graph of cell cycle-related proteins in bladder cancer cells (A: Expression of cell cycle-related proteins in 5637 cells; B: Statistical graph of cell cycle-related proteins in 5637 cells; C: Expression of cell cycle-related proteins in T24 cells; D: Statistical graph of cell cycle-related proteins in T24 cells). Figure 10 : Expression and statistical graph of apoptosis-related proteins in bladder cancer cells (A: Expression of 5637 apoptosis-related proteins; B: Statistical graph of 5637 apoptosis proteins; C: Expression of T24 apoptosis-related proteins; D: Statistical graph of 5637 apoptosis proteins). Figure 11 : Expression and statistical graph of pathway-related proteins in bladder cancer cells (A: Expression of proteins related to the 5637 pathway; B: Statistical graph of proteins related to the 5637 pathway; C: Expression of proteins related to the T24 pathway; D: Statistical graph of proteins related to the T24 pathway). Detailed Implementation

[0017] This invention does not specifically limit the source of paclitaxel flavonoids; they can be synthesized using conventional chemical synthesis methods in the art. Pyracalitaxel flavonoids have good prepareability and stability and can also be purchased commercially. The following examples further illustrate this invention, but should not be construed as limiting the scope of the invention.

[0018] Example 1: Activity of Taxodium flavonoids on bladder cancer cells 5637 and T24 After thoroughly mixing the cells from the culture flask, prepare a single-cell suspension. Transfer the suspension to 15 ml centrifuge tubes using a pipette, centrifuge at 800 rpm for 5 min, discard the supernatant, add an appropriate amount of PBS for washing, centrifuge at 800 rpm for 5 min, discard the PBS, and repeat the washing process 3 times. Resuspend the cells in fresh 1640 complete culture medium, mix thoroughly, and use a 10 μL pipette tip to aspirate the single-cell suspension for cell counting. After counting, collect the cells at 100 μL / well (5 × 10⁶ cells / well). 3 Inoculate 0, 30, 60, 90, 120, and 150 μM of paclitaxel flavonoids into each well of a 96-well plate with six replicates. Incubate at 37°C and 5% CO2 for 1-5 days. Then, add 10 μL of CCK-8 solution to each well and incubate for 2-4 hours. Remove the 96-well plate, measure the absorbance using a microplate reader, and plot a line graph.

[0019] IC50 was calculated using GraphPad Prism 8.3.1, and the low, medium, and high concentrations used in subsequent experiments were determined based on the IC50 results. The results are as follows: Figure 1 As shown, the inhibitory effects of paclitaxel flavonoids on bladder cancer cells (5637 and T24) were time- and concentration-dependent, with 24-hour IC50 values ​​of 104.27±5.71 μm and 109.12±5.58 μm, respectively. Figure 1 Therefore, in this experiment, 0 μm was set as the control group, and the concentrations of Taxus chinensis flavonoids of 50 μm, 100 μm, and 150 μm were set as the low, medium, and high concentration groups of the experimental group, and 24 hours was set as the time intervention condition.

[0020] Example 2: Morphological changes of bladder cancer cells 5637 and T24 by cypermethrin. Bladder cancer cells 5637 and T24 were seeded into 24-well plates at a cell number of 1×10⁶. 4 Cells were divided into experimental groups (50, 100, 150 μm) and a control group (0 μm) based on CCK-8 results, with three replicates per group. Once cells had fully adhered and reached 60% confluence, Selaginella extract was added and cultured for 24 hours. Cells were stained with 1% crystal violet and observed under a microscope to assess cell growth. Microscopic observation of bladder cancer 5637 and T24 cells after treatment with paclitaxel showed that with increasing paclitaxel concentration, cell number gradually decreased, cells atrophied, and varying degrees of nuclear pyknosis occurred. Figure 2 ).

[0021] Example 3: EDU experiment to detect the proliferation of bladder cancer 5637 and T24 cells after treatment with paclitaxel flavonoids. Bladder cancer cells 5637 and T24 were seeded into 24-well plates at a cell number of 1 × 10⁻⁶. 4 Cells were cultured at 10 mg / well until fully adhered, then drug was added according to the experimental groups, and cultured at 37°C for 24 h. The old culture medium was then removed, and fresh culture medium containing 10 mg / L EdU / Hoechst 33342 fluorescent dye was added. The cells were then incubated in a 5% CO2, 37°C cell culture incubator for 2 h. Finally, the cells were observed and imaged under a fluorescence microscope. EDU assays showed that, compared with the control group, cell proliferation rate decreased significantly after drug addition. In the T24 cell line, the proliferation rate was 83% in the control group, 64% in the low-concentration group, 48% in the medium-concentration group, and 34% in the high-concentration group. The high-concentration group showed a 49% decrease in proliferation rate compared to the control group. In the 5637 cell line, the proliferation rate was 79% in the control group, 60% in the low-concentration group, 45% in the medium-concentration group, and 35% in the high-concentration group. The high-concentration group showed a 44% decrease in proliferation rate compared to the control group. Compared with the control group, cell proliferation was significantly reduced after the addition of cypermethrin. Figure 3 These results indicate that paclitaxel flavonoids inhibit the proliferation of bladder cancer cells.

[0022] Example 4: Effects of Taxodium flavonoids on the cell cycle of bladder cancer cells 5637 and T24 cells were treated with 0, 50, 100, and 150 μM of paclitaxel flavonoids for 24 hours. Cells were resuspended in sterile PBS and centrifuged for 5 min (800 rpm / min), repeated twice, to remove trypsin. Cells were collected at 1×10⁻⁶ m². 6 Cells were then slowly fixed with pre-cooled 70% ethanol and stored overnight at 4°C. The next day, bladder cancer cells were washed twice with pre-cooled PBS solution, centrifuged for 5 min (800 pm / min), and the cell pellet after removing the fixative was collected. Following the cell cycle kit instructions, 100 μl of RNase A (25 μg / ml) and 400 μl of propidium iodide dye (50 μg / ml) were added, and the cells were incubated at 4°C in the dark for 40 min. After incubation, the cells were analyzed using flow cytometry, and the data were analyzed and visualized using FlowJo 10.6.2 software. The results are as follows: Figure 4As shown, in the 5637 cell line, compared with the control group, the number of cells in S phase increased by 4.7%, 13.6%, and 20.6% in the low, medium, and high concentration groups, respectively; in the T24 cell line, compared with the control group, the number of cells in S phase increased by 5.4%, 7.3%, and 18.2% in the low, medium, and high concentration groups, respectively. With increasing concentration of paclitaxel flavonoids, the number of cells in S phase decreased. Furthermore, we used Western blotting to measure the changes in the expression of related proteins in bladder cancer cells 24 hours after treatment with paclitaxel flavonoids. With increasing paclitaxel flavonoid concentration, the expression of c-Myc and cyclin D1 was significantly downregulated (…). Figure 9 These data indicate that paclitaxel flavonoids can arrest the cell cycle of bladder cancer cells, thereby affecting their proliferation.

[0023] Example 5: Effect of Taxodium flavonoids on apoptosis of bladder cancer cells 5637 and T24 cells were treated with 0, 50, 100, and 150 μM of paclitaxel flavonoids for 24 hours. Cells were resuspended in sterile PBS and centrifuged for 5 min (800 rpm / min), repeated twice, to remove trypsin. Cells were collected (1 × 10⁻⁶ cells / min). 6 Cells were collected according to the instructions of the apoptosis detection kit. 500 μl of binding buffer was added to each sample EP tube to prepare a cell suspension (gentle pipetting was required). Annexin V-FITC / PI fluorescent dye was then added, and the cells were incubated at room temperature in the dark for 15 minutes. After thorough mixing, the suspension was analyzed using FlowJo 10.6.2 software. Western blotting was used to detect the expression of apoptosis-related proteins BCl2 and Bax. The results showed that in the T24 cell line, compared with the control group, the apoptosis rate was 4.08% at low concentration, 8.65% at medium concentration, and 13.34% at high concentration. In the 5637 cell line, compared with the control group, the apoptosis rate was 8.61% at low concentration, 9.43% at medium concentration, and 13.26% at high concentration. Compared with the control group, the percentage of apoptosis in both 5637 and T24 cells significantly increased with increasing concentration of Taxodium styracifolium flavonoids. Figure 5 AC). Western blot analysis showed that with increasing concentrations of paclitaxel flavonoids, the expression level of Bcl-2 in bladder cancer cells gradually decreased, while the expression level of Bax was significantly higher than that in the control group (AC). Figure 10 These data indicate that paclitaxel flavonoids can induce apoptosis in bladder cancer cells.

[0024] Example 6: Cell migration ability of bladder cancer 5637 cells and T24 cells after treatment with paclitaxel flavonoids Bladder cancer cells 5637 and T24 were seeded into 6-well plates at a density of 4 × 10⁶ cells / well. 5 Cells were cultured in 6-well plates, and after they adhered and grew to 90% of the total area, they were scratched perpendicularly to the marked line on the back of the well using a 10 μL pipette tip. Exfoliated cells were washed away with PBS, and the cells were observed and photographed under a microscope. Taxone was then added, and control (0 μm) and experimental groups (50, 100, and 150 μm) were set up. Serum-free medium was added and the cells were cultured for 24 hours. The healing of the scratches was observed and images were taken under an inverted microscope. The experiment was repeated three times, and cell migration rate was calculated. The results showed that the migration rate of the control group was significantly higher than that of the experimental group. In the 5637 cell line, the migration rate of the control group was 71.7%, the low concentration group was 48%, the medium concentration group was 24.3%, and the high concentration group was 14%. In the T24 cell line, the migration rate of the control group was 75%, the low concentration group was 54%, the medium concentration group was 30.7%, and the high concentration group was 16.7%. This indicates that taurine flavonoids can inhibit the migration of bladder cancer cells 5637 and T24, and the migration rate decreases with increasing drug concentration. Figure 6 ).

[0025] Example 7: Effects of Taxodium flavonoids on the cell invasion ability of bladder cancer 5637 cells and T24 cells Before the experiment, prepare pre-chilled sterile pipette tips. Use the pre-chilled tips to mix Matrigel into a homogenous paste. Add pre-chilled serum-free fresh culture medium at a ratio of 1:7 and mix well. Spread the diluted Matrigel evenly in the upper chamber of the chamber and incubate at 37°C for 30 minutes. After gel formation, proceed with subsequent operations. (The last part, "1×10," appears to be an unrelated instruction and is left untranslated.) 5 Two hundred and ten cells (200 µl) were placed in a Transwell chamber (8 µm, Corning, USA), and different concentrations of paclitaxel flavonoids (50, 100, and 150 μm) were added as required for the experiment. 600 µl of 1640 medium containing 10% fetal bovine serum was added to the lower compartment. After 24 hours, the cells in the upper compartment were removed. Cell invasion was observed and images were taken under an inverted microscope. The results showed that, compared with the control group, the invasive ability of 5637 cells and T24 cells was significantly reduced after the addition of paclitaxel flavonoids (50, 100, and 150 μm). p < 0.01)( Figure 7 ).

[0026] Example 8: Western blot analysis and immunofluorescence assay to determine whether the effect of paclitaxel on bladder cancer 5637 cells and T24 cells is related to the PI3K / Akt / NF-κB pathway. We analyzed the localization of PI3K using immunofluorescence and detected the effect of paclitaxel flavonoids using Western blotting. Expression of PI3K, Akt, and NF-κB proteins, which are related to the PI3K / Akt / NF-κB pathway. The results showed that... Compared with the blank control group, the low, medium, and high dose groups of Taxodium styracifolium flavonoids showed blue fluorescence after DAPI staining. Cell nuclei; green fluorescence indicates PI3K. PI3K expression in the nucleus decreased after treatment with paclitaxel. ( Figure 8 Western blotting results showed that with increasing concentrations of paclitaxel flavonoids, the protein expression of PI3K, AKT, and NF-κB significantly decreased. Figure 11 These findings suggest that the PI3K / AKT / NF-κB pathway is involved in the apoptosis induced by paclitaxel flavonoids in bladder cancer cells.

[0027] Based on the above experimental examples, Taxus chinensis flavonoids can promote apoptosis in bladder cancer (5637 and T24) and inhibit the proliferation, migration and invasion of 5637 and T24 cells by inducing S phase arrest. This process is related to the PI3K / Akt / NF-κB pathway.

Claims

1. Use of amentoflavone in the preparation of a medicament for treating bladder cancer.

2. The use of amentoflavone according to claim 1 for the preparation of a medicament for the treatment of bladder cancer, characterized in that: The amentoflavone has the following structural formula: 。 3. The use of amentoflavone according to claim 1 for the preparation of a medicament for the treatment of bladder cancer, characterized in that: The amentoflavone inhibits the viability, proliferation, invasion and migration of bladder cancer cells.

4. The use of amentoflavone according to claim 1 for the preparation of a medicament for the treatment of bladder cancer, characterized in that: The amentoflavone induces apoptosis of bladder cancer cells by promoting the expression of pro-apoptotic gene Bax and inhibiting the expression of apoptosis-inhibiting gene Bcl-2.

5. Use of the fisetinidol according to claim 1 for the preparation of a medicament for the treatment of bladder cancer, characterized in that: The amentoflavone inhibits the proliferation of bladder cancer cells by arresting them in the G1 phase and promotes the expression of cell cycle inhibitory protein p21.

6. Use of the fisetinidol according to claim 1 for the preparation of a medicament for the treatment of bladder cancer. The amentoflavone induces the apoptosis of bladder cancer cells by inducing the reduction of the expression of PI3K, AKT and NF-κB proteins in the nucleus through the PI3K / AKT / NF-κB pathway.

7. Use of amentoflavone according to any one of claims 1 to 6 for the preparation of a medicament for the treatment of bladder cancer, characterized in that: The bladder cancer cells are bladder cancer T24 cells and bladder cancer 5637 cells.

8. The use of the fisetin according to claim 1 for the preparation of a medicament for the treatment of bladder cancer, characterized in that: The medicament is prepared by adding amentoflavone as the only active ingredient into a pharmaceutically acceptable carrier and / or excipient to form a pharmaceutically acceptable dosage form.

9. Use of the compound of the formula (I) according to claim 8 for the preparation of a medicament for the treatment of bladder cancer. The dosage form is a tablet, a capsule, a granule, an oral solution, a suspension or a needle.

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