Application of tripterine in preparation of bladder cancer resisting medicine

Abstract

The invention discloses a new application of tripterine, namely application of the tripterine in preparation of bladder cancer resisting drugs, in-vitro cell experiments prove that the tripterine shows remarkable proliferation inhibition activity on various bladder cancer cell strains including cell strains UM-UC-3, TCCSUP and T24 and a cis-platinum drug-resistant strain T24CDDP, the median inhibitory concentration (IC50) of the tripterine is 0.2688-0.5870 mu M, and the tripterine shows remarkable proliferation inhibition activity on various bladder cancer cell strains including cell strains UM-UC-3, TCCSUP and T24 and a cis-platinum drug-resistant strain T24CDDP. The further mechanism research shows that the tripterine can regulate and control the cell cycle by down-regulating the expression levels of CDK1 and CDC5L proteins in bladder cancer cells and up-regulating the expression of phosphorylated p53 (p-p53) proteins at the same time, thereby playing a role in resisting bladder cancer; the invention provides a new effective candidate drug for clinical treatment of bladder cancer, especially cis-platinum resistant bladder cancer, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of triptolide in the preparation of anti-bladder cancer drugs, and particularly to anti-bladder cancer drugs and related preparations using triptolide as the active ingredient to inhibit the proliferation of bladder cancer cells, induce apoptosis of bladder cancer cells, or arrest the cell cycle of bladder cancer cells. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system worldwide, ranking first in incidence among urinary system malignancies, and its incidence has been rising continuously in recent years. Based on pathological type, bladder cancer is mainly divided into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Non-muscle-invasive bladder cancer accounts for approximately 70%-80% of cases, but its postoperative recurrence rate is as high as 50%-70%. Some patients may also progress to muscle-invasive bladder cancer, seriously threatening their lives and health.

[0003] Currently, clinical treatments for bladder cancer mainly include surgery, chemotherapy, radiotherapy, and immunotherapy. For non-muscle-invasive bladder cancer, transurethral resection of bladder tumor (TURBT) is the preferred treatment, but postoperative intravesical instillation therapy is necessary to reduce the recurrence rate. Commonly used instillation drugs include BCG, epirubicin, and pirarubicin. For muscle-invasive bladder cancer, radical cystectomy combined with pelvic lymph node dissection is usually performed, followed by platinum-based systemic chemotherapy. However, existing treatment regimens have significant limitations: on the one hand, intravesical instillation therapy easily causes bladder irritation symptoms such as urinary frequency, urgency, and dysuria, and some patients do not respond to drugs such as BCG or develop drug resistance, leading to treatment failure; on the other hand, systemic chemotherapy drugs have strong toxic side effects, easily causing bone marrow suppression, gastrointestinal reactions, and liver and kidney damage, and long-term use easily leads to multidrug resistance, resulting in decreased treatment efficacy. Therefore, finding novel anti-bladder cancer drugs that are widely available, have high safety profiles, clear mechanisms of action, and can effectively inhibit the progression of bladder cancer has become a key issue that urgently needs to be addressed in the current field of bladder cancer treatment.

[0004] Celastrol, also known as tripterygium sorbitol, is derived from the Celastraceae plant Tripterygium wilfordii (Tripterygium wilfordii). Tripterygium wilfordiiTripterygium wilfordii is a pentacyclic triterpenoid compound extracted and isolated from the root bark of *Hook. f.*. Existing research indicates that triptolide possesses various pharmacological activities, including anti-inflammatory, immunosuppressive, and antitumor effects. It has shown certain antitumor activity in various malignant tumor models, including lung cancer, breast cancer, liver cancer, and colorectal cancer. Its mechanisms of action mainly include inhibiting tumor cell proliferation, inducing tumor cell apoptosis, arresting the tumor cell cycle, inhibiting tumor angiogenesis, and regulating tumor-related signaling pathways (such as NF-κB, STAT3, and PI3K / Akt). However, prior to this application, no published literature or patents have reported that triptolide has a specific inhibitory effect on bladder cancer, nor has any application of triptolide in the preparation of anti-bladder cancer drugs been discussed. Summary of the Invention

[0005] In response to the problems of significant side effects, strong drug resistance, and limited therapeutic effects of existing bladder cancer treatments, this invention provides the application of triptolide in the preparation of anti-bladder cancer drugs. Experiments have demonstrated that triptolide can significantly inhibit the proliferation of bladder cancer cells, induce apoptosis in bladder cancer cells, and arrest the cell cycle of bladder cancer cells.

[0006] The active ingredient (or active ingredient) of the drug of the present invention is triptolide, and one or more pharmaceutically acceptable excipients may be added, or it may be compounded with other active ingredients to exert an inhibitory effect; in addition to being made into tablets, the preparation may also be made into various pharmaceutically acceptable forms such as pills, powders, capsules, granules, oral liquids and injections.

[0007] This invention, through MTT cell proliferation assays, confirms that triptolide can significantly inhibit the proliferation of bladder cancer cells. Triptolide also showed inhibitory effects on the IC50 of T24, T24CDDP, UM-UC-3, and TCCSUP cells. 50 The values ​​ranged from 0.2688 μM to 0.5870 μM. Western blotting experiments revealed that triptolide downregulated the expression levels of CDK1 and CDC5L proteins and upregulated the expression level of phosphorylated p53 protein in bladder cancer cells. Triptolide reduced the proliferation capacity of bladder cancer cells by arresting cell cycle progression. Flow cytometry experiments showed that triptolide significantly promoted apoptosis in bladder cancer cells. This invention provides a new and effective candidate drug for the clinical treatment of bladder cancer, especially cisplatin-resistant bladder cancer, and has important clinical application value. Attached Figure Description

[0008] Figure 1 The IC50 of triptolide against human bladder cancer cells T24 and T24CDDP in the MTT cell proliferation assay. 50 Value results; the left figure shows the IC50 value of triptolide on T24 cells.50 The right figure shows the IC50 value of triptolide on T24CDDP. 50 value; Figure 2 The IC50 of triptolide against human bladder cancer cells UM-UC-3 and TCCSUP in the MTT cell proliferation assay. 50 Results; the left figure shows the IC50 value of triptolide on UM-UC-3 cells. 50 The right figure shows the IC50 value of triptolide on TCCSUP. 50 value; Figure 3 The images show the results of Western blotting of T24CDDP cells treated with different concentrations of triptolide. The top image is an electrophoresis diagram, and the bottom image is a statistical graph. Figure 4 The images show the results of Western blotting of UM-UC-3 cells treated with different concentrations of triptolide. The top image is an electrophoresis diagram, and the bottom image is a statistical graph. Figure 5 This is the flow cytometry result of triptolide's effect on apoptosis in human bladder cancer cells T24CDDP. Figure 6 This is a statistical chart showing the detection results of triptolide on apoptosis in human bladder cancer cells T24CDDP. Figure 7 The results are from flow cytometry analysis of the effects of triptolide on apoptosis in human bladder cancer cells UM-UC-3. Figure 8 This is a statistical graph showing the detection results of triptolide on apoptosis in human bladder cancer cells UM-UC-3. Detailed Implementation

[0009] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments, but these embodiments should not be used to interpret the limitation of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available. After reading this description, any equivalent modifications, alterations, and modifications made by those skilled in the art are within the scope defined by the claims of the present invention.

[0010] Reagents: RPMI 1640 medium (Gibco, USA), fetal bovine serum (Guangzhou Saiku Biotechnology Co., Ltd.), trypsin (Gibco, USA), Opti-MEM medium (Gibco, USA).

[0011] Cell lines: Bladder cancer cell lines T24, UM-UC-3, and TCCSUP, purchased from Guangzhou Saiku Biotechnology Co., Ltd.

[0012] Example 1: Inhibitory effect of triptolide on the proliferation of bladder cancer 1. Preparation of cisplatin (CDDP) resistant strain of human bladder cancer cell line T24 Human bladder cancer T24 cells were seeded into T25 culture flasks containing high-glucose DMEM medium with 10% fetal bovine serum and placed in a cell culture incubator at 37°C and 5% CO2. Cisplatin-resistant cells were induced by gradually increasing the drug dose, using 0.5 μM CDDP as the initial concentration. After the cells were cultured until they could grow stably and were passaged 3 times, the CDDP concentration was gradually increased until the cells could grow stably in the culture medium with a CDDP concentration of 10 μM and were successfully passaged 10 times. This obtained a cisplatin-resistant (CDDP) human bladder cancer cell model, which was named cisplatin-resistant human bladder cancer cell (T24CDDP cells).

[0013] 2. Bladder cancer cell lines T24, T24CDDP, UM-UC-3, and TCCSUP were evenly seeded into 96-well plates (4000 cells / well, 100 μL per well) and incubated at 37°C and 5% CO2 for 24 hours. After complete cell adhesion, the cells were treated with different concentrations (1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM) of triptolide (CST) for 48 hours. Then, 30 μL of MTT solution (5 mg / mL) was added to the 96-well plates, and the plates were incubated at 37°C and 5% CO2 for 5 hours. The liquid in the plates was then discarded, and 100 μL of [the solution] was added to the plates. DMSO solution (99.99% purity) was used, shaken on a shaker for 10 min (200 rpm), and the absorbance was measured at 570 nm. Cell viability was calculated, a dose-response curve was plotted, and the IC50 was calculated. 50 value; See results Figure 1-2 As shown in the figure, triptolide inhibited the proliferation of bladder cancer cells in a concentration-dependent manner. The IC50 values ​​of triptolide on T24, T24CDDP, UM-UC-3, and TCCSUP cells were also observed. 50 The values ​​were 0.2688 μM, 0.5366 μM, 0.5870 μM, and 0.3872 μM, respectively, indicating that triptolide can significantly inhibit the proliferation of bladder cancer cells.

[0014] Example 2: Tripterygium wilfordii can arrest the cell cycle of bladder cancer cells. T24CDDP and UM-UC-3 bladder cancer cells in logarithmic growth phase were evenly seeded into 6-well plates and placed in a cell culture incubator. The cells were cultured at 37°C and 5% CO2 for 24 hours. After the cells were completely adhered, they were treated with different concentrations (1 μM, 2 μM, 4 μM) of CST for 24 hours. The total protein of the cells was then extracted and Western blotting was performed to detect the expression levels of CDK1, CDC5L, p-p53, and GAPDH proteins. See results Figure 3-4 As can be seen from the figure, triptolide downregulated the levels of CDK1 and CDC5L proteins and upregulated the level of p-p53 protein in a concentration-dependent manner, suggesting that triptolide can arrest the cell cycle process and reduce the proliferation ability of bladder cancer cells.

[0015] Example 3: Tripterygium wilfordii-induced apoptosis in bladder cancer cells T24CDDP and UM-UC-3 bladder cancer cells in logarithmic growth phase were evenly seeded into 6-well plates and cultured at 37°C and 5% CO2 for 24 hours. After complete cell adhesion, the cells were treated with different concentrations (1 μM, 2 μM, and 4 μM) of CST for 24 hours, followed by gentle digestion with 0.25% trypsin (without EDTA) to avoid over-digestion and false positives. The trypsin was neutralized with serum-containing medium, centrifuged at 1000 rpm for 5 min, washed twice with PBS, and resuspended in PBS. 100 μL of the cell suspension (approximately 1 × 10⁻⁶ cells / well) was collected. 5 Add cells to a flow cytometry tube, add 5 μL Annexin V-FITC, mix gently, incubate at room temperature in the dark for 15 min, add 5 μL PI (final concentration 1-2 μg / mL), incubate in the dark for 5 min, and immediately add 400 μL Binding Buffer. Avoid prolonged storage (test within 1 hour).

[0016] See results Figure 5-8 As can be seen from the figure, triptolide can significantly promote apoptosis of bladder cancer cells. Flow cytometry analysis revealed that triptolide inhibits the proliferation of bladder cancer cells in a concentration-dependent manner and induces apoptosis.

[0017] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be covered by the present invention.

Claims

1. Application of celadixyn in the preparation of drugs for resisting bladder cancer.

2. Use according to claim 1, characterized in that: The half-inhibitory concentration of celadixyn on bladder cancer cell lines UM-UC-3, TCCSUP, T24 and T24CDDP is 0.2688-0.5870 μM.

3. The use according to claim 1, characterized in that: Celadixyn achieves the purpose of resisting bladder cancer by down-regulating the expression level of CDK1 protein and CDC5L protein in bladder cancer cells and up-regulating the expression level of phosphorylated p53 protein in bladder cancer cells.

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