Application of polyphyllin VII in preparation of bladder cancer chemotherapy sensitizer
By combining Paris polyphylla saponin VII with cisplatin, the Akt-SREBP1 lipid metabolism pathway was inhibited, solving the chemotherapy resistance of bladder cancer and achieving a significant improvement in chemotherapy effect and enhanced safety.
Patent Information
- Application Number
- CN202511186110.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-23
- Publication Date
- 2025-10-17
AI Technical Summary
In the existing technology, the problem of chemotherapy resistance in bladder cancer, especially resistance to cisplatin, has not been effectively solved. Abnormal activation of the Akt-SREBP1 lipid metabolism pathway is a key factor. Existing chemotherapy sensitizers have poor targeting and large toxic side effects.
Paris polyphylla saponin VII (PPVII) is used in combination with cisplatin to inhibit the Akt-SREBP1 lipid metabolism pathway, regulate the expression of key proteins, enhance the chemotherapy effect, and reduce drug resistance.
It significantly improves the sensitivity of bladder cancer chemotherapy, reduces the dosage of cisplatin, enhances the effect of chemotherapy, and has no obvious toxic side effects, providing a safe and efficient sensitization solution.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of biological medicine, and particularly relates to application of Paris saponin VII in preparation of a bladder cancer chemotherapy sensitizer. BACKGROUND
[0002] Bladder cancer is one of the most common malignant tumors of the urinary system worldwide. For muscle-invasive or metastatic bladder cancer, platinum-based combination chemotherapy is the standard treatment regimen, in which cisplatin is the core drug. However, primary or secondary cisplatin resistance is the main cause of bladder chemotherapy failure and even patient death. Therefore, in-depth elucidation of the intrinsic molecular mechanism of cisplatin resistance and search for effective sensitization strategies are key scientific problems to be solved to improve the treatment effect of bladder cancer.
[0003] Recent studies have shown that metabolic reprogramming of tumor cells is not only one of the hallmark features of cancer, but also a key factor driving chemotherapy resistance. Among them, abnormal activation of lipid metabolism plays a crucial role in maintaining tumor cell survival, proliferation, invasion and drug resistance. Sterol regulatory element binding protein 1 (SREBP1) as a core transcription factor regulating lipid synthesis and metabolism, its activity is precisely regulated by the upstream Akt (protein kinase B) signaling pathway. Activated Akt (p-Akt) activates SREBP1 through multiple mechanisms, and then drives the excessive synthesis of lipids such as fatty acids and cholesterol. These lipids provide rich biomembrane building materials, energy sources and signaling molecules for drug-resistant tumor cells, thereby enhancing their survival ability and weakening the killing effect of chemotherapy drugs. Existing technologies show that abnormal activation of the Akt-SREBP1 signaling pathway is closely related to the chemotherapy resistance of various solid tumors. However, the specific role of this pathway in cisplatin resistance of bladder cancer and whether it can be used as a target to reverse drug resistance remain to be further explored.
[0004] At present, although some studies have attempted to use broad-spectrum Akt inhibitors to overcome chemotherapy resistance, they often have problems such as poor selectivity, large toxic side effects, and limited clinical application. Therefore, finding new active ingredients from natural products that can precisely and safely intervene in specific drug resistance pathways has become a hot research topic. Paris saponin VII is an active steroidal saponin extracted from the traditional Chinese medicine Paris, and previous studies have reported that it has broad anti-tumor activity. However, whether it can reverse cisplatin resistance of bladder cancer by regulating lipid metabolism pathways has not been reported.
[0005] In summary, there is an urgent need in the art for a new drug or regimen that can effectively inhibit the Akt-SREBP1 lipid metabolism pathway and thus overcome cisplatin resistance of bladder cancer. SUMMARY
[0006] The application provides a new use of rhizoma paridis saponin VII (PP VII), that is, application of the rhizoma paridis saponin VII in preparation of a bladder cancer chemotherapy sensitizer.
[0007] The application also provides a medicine composition, that is, the rhizoma paridis saponin VII and cisplatin are used for preparation of a medicine for treating bladder cancer, and the rhizoma paridis saponin VII overcomes cisplatin drug resistance by inhibiting an Akt-SREBP1 lipid metabolism pathway.
[0008] The chemical structural formula of the rhizoma paridis saponin VII is as follows: .
[0009] The application proves that the PP VII can significantly inhibit expression of key proteins p-PI3K, p-AKT and p-mTOR in an Akt signal pathway, up-regulate expression levels of apoptosis markers c-PARP and c-cas-7, and increase an expression amount of a cell cycle control protein P21 by Western blot detection. Then, synergistic effects of the two drugs are evaluated through in-vitro and in-vivo experiments, in-vitro experiments prove that the PP VII can significantly improve an apoptosis rate of T24 CDDP drug-resistant cells, and the PP VII and the cisplatin have a synergistic effect. In-vivo experiments prove that the PP VII single-drug treatment shows a significant tumor inhibition effect, and the PP VII and the cisplatin have a strong synergistic effect, a CDI of the PP VII and the cisplatin is 0.45, and the PP VII and the cisplatin do not cause obvious toxic side effects.
[0010] The PP VII plays a sensitization role through the following mechanisms: (1) dose-dependent inhibition of Akt pathway activity, which is manifested as that p-PI3K, p-AKT, p-mTOR, p-GSK3beta and p-P70S6K levels are reduced; (2) up-regulation of P21 protein expression; (3) inhibition of expression of SREBP1, ACC, FASN and SCD1 which are components of an SREBP1 signal pathway; (4) up-regulation of expression of apoptosis markers c-PARP and c-cas-7.
[0011] The application discloses a medicine composition (or an effective component) of the rhizoma paridis saponin VII, and one or more pharmaceutically acceptable adjuvants can be added, or the rhizoma paridis saponin VII is compounded with other active components to play an inhibitory role.
[0012] Compared with the prior art, the application has the following advantages: The application first systematically proves that PPVII plays an anti-tumor role by regulating the Akt signaling pathway. The influence of PPVII on the apoptosis markers and cell cycle proteins is determined, and a complete pharmacodynamic evaluation system is established. The synergistic effect is significant: the combination of PPVII and cisplatin produces a strong synergistic effect (CDI=0.45), which can significantly reduce the dosage of cisplatin and improve the treatment effect of drug-resistant tumors. The safety is outstanding: no obvious toxicity is observed at the therapeutic dose, and the toxicity and side effects of cisplatin are not aggravated, which has good clinical application safety and provides a new solution to overcome tumor drug resistance. The application solves the key technical problems of poor targeting and large toxicity and side effects of the existing chemosensitizers by innovatively discovering the mechanism of PPVII in overcoming cisplatin resistance of bladder cancer, and has important clinical application value. BRIEF DESCRIPTION OF DRAWINGS
[0013] Figure 1 is the experimental result of PPVI inhibiting the Akt signaling pathway of T24CDDP cells in a dose-dependent manner. The upper graph is a protein electrophoresis graph, and the lower graph is a statistical graph of the expression amount; Figure 2 is the experimental result of PPVII down-regulating the key components of the SREBP1 signaling pathway of T24CDDP cells. The upper graph is a Western blotting graph, and the lower graph is a statistical graph of the expression amount; Figure 3 is the T24CDDP cell apoptosis graph detected by flow cytometry of PPVII on T24CDDP cell apoptosis; Figure 4 is the T24CDDP cell apoptosis graph detected by flow cytometry of PPVII on T24CDDP cell apoptosis; Figure 5 is the statistical result graph of the flow cytometry detection result of PPVII on T24CDDP cell apoptosis; Figure 6 is the Western blotting result of T24CDDP treated with different concentrations of PPVII. The left graph is an electrophoresis graph, and the middle and right graphs are statistical graphs; Figure 7 is the IC 50 value result of the MTT cell proliferation experiment of PPVII on T24CDDP cells; Figure 8 is the inhibition effect graph of PPVII single drug, cisplatin single drug and the combination of the two drugs on the volume and weight of T24CDDP transplanted tumors. The left graph is a tumor size schematic diagram, the middle graph is a tumor volume statistical graph, and the right graph is a tumor weight statistical graph; Figure 9 is the statistical graph of the influence of PPVII single drug, cisplatin single drug and the combination of the two drugs on the body weight of experimental animals; Figure 10 is the influence of PPVII single drug, cisplatin single drug and the combination of the two drugs on the morphology of T24CDDP transplanted tumor tissues. DETAILED DESCRIPTION
[0014] The technical solutions of the present application will be further described below in combination with the drawings and specific embodiments, but these embodiments shall not be used to explain the limitation of the present application. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified; after reading the description of the present application, various modifications, changes and modifications of the equivalents thereof by the skilled person in the art all belong to the scope defined by the claims of the present application.
[0015] Reagents: MTT reagent (Solarbio, Beijing, China), RPMI 1640 medium (Guangzhou Sike Biotechnology Co., Ltd.), MEM medium (Guangzhou Sike Biotechnology Co., Ltd.), fetal bovine serum (Guangzhou Sike Biotechnology Co., Ltd.), trypsin (Gibco, USA), JC-10 detection kit (Jiangsu Kaiji Biotechnology Co., Ltd.), apoptosis detection kit (Jiangsu Kaiji Biotechnology Co., Ltd.).
[0016] Cell line: human bladder cancer T24 cells, purchased from Guangzhou Sike Biotechnology Co., Ltd.
[0017] Example 1: Detection of the regulation of PPⅦ on key signal proteins of T24CDDP cells 1. Preparation of cisplatin (CDDP) resistant strain of human bladder cancer cell T24 Human bladder cancer T24 cells were inoculated in T25 culture bottles containing 10% fetal bovine serum in high glucose DMEM medium and placed in a cell culture incubator for culture at 37℃ and 5% CO2. Cisplatin-resistant cells were induced by gradually increasing the drug dose, using 0.5 μM / L of CDDP as the initial concentration. After the cells were stably grown and continued to be cultured and passaged for 3 times, the CDDP concentration was gradually increased until the cells could stably grow and be smoothly passaged in the culture medium with CDDP concentration of 10 μM / L. After 20 times of passage, a cisplatin-resistant human bladder cancer cell model was obtained, which was named as cisplatin-resistant human bladder cancer cell (T24CDDP cell).
[0018] 2. The logarithmically growing T24CDDP cells were inoculated in 6-well plates and placed in a cell culture incubator for culture at 37℃ and 5% CO2 for 24 hours. After the cells were completely adherent, the cells were treated with PPⅦ at concentrations of 2 μM / L and 4 μM / L for 24 hours, respectively. Then the total protein of the cells was extracted for Western blotting experiment to detect the expression levels of c-PARP, c-cas-7, P21, p-PI3K, p-Akt, p-mTOR, p-GSK3β, p-P70S6K, SREBP1, ACC, FASN and SCD1 proteins. A control group without addition of PPⅦ was also set. Results are shown in Figures 1-2, from which Figure 1 it can be seen that, compared with the control group, PPVII up-regulates the expression of c-PARP, c-cas-7 and P21 in T24 CDDP cells, while down-regulating the levels of p-PI3K, p-Akt, p-mTOR and p-GSK3β.
[0019] From Figure 2 it can be seen that, compared with the control group, PPVII reduces the expression of p-P70S6K, SREBP1, ACC, FASN and SCD1 in T24 CDDP cells.
[0020] The above results show that PPVII inhibits the activity of the Akt pathway in a dose-dependent manner (as shown by the reduced phosphorylation levels of p-PI3K, p-AKT, p396 mTOR, p-GSK3β and p-P70S6K, accompanied by increased P21 expression), while also inhibiting components of the SREBP1 signaling pathway (SREBP1, ACC, FASN and SCD1).
[0021] Example 2: Apoptosis analysis (1) T24 CDDP cells in the logarithmic growth phase were evenly inoculated in a 6-well plate and placed in a cell culture incubator for 24 hours at 37°C, 5% CO2. After the cells were completely adherent, they were treated with PPVII (2 μM / L, 4 μM / L), CDDP (50 μΜ / L), SC79 (20 μΜ / L), PPVII (4 μM / L) + CDDP (50 μΜ / L), PPVII (4 μM / L) + SC79 (20 μΜ / L), and PPVII (4 μM / L) + CDDP (50 μΜ / L) + SC79 (20 μΜ / L) for 24 hours. Then, the cells were gently digested with 0.25% trypsin (without EDTA) to avoid false positives caused by excessive digestion. The trypsin was neutralized with serum-containing medium, and the cells were centrifuged at 1000 rpm for 5 minutes and washed twice with PBS. The cells were resuspended in PBS, and 100 μL of cell suspension (about 1 x 10 5 Cells) was added to a flow tube. 5 μL of Annexin V-FITC was added, and the mixture was gently mixed. The mixture was incubated at room temperature for 15 minutes in the dark. 5 μL of PI (final concentration 1-2 μg / mL) was added, and the mixture was incubated for 5 minutes in the dark. 400 μL of Binding Buffer was immediately added to avoid long-term storage (within 1 hour of detection). A blank control group without the addition of agents was also set up. The flow cytometry detection results are shown in Figures 3-5. As can be seen from the figures, compared with the blank control group, PP VII (4 μM / L) and CDDP (50 μΜ / L) can promote the apoptosis of cisplatin-resistant bladder cancer cells. Flow cytometry detection found that the PP VII treatment group can inhibit the proliferation of bladder cancer cells in a concentration-dependent manner and induce cell apoptosis. After adding the apoptosis inhibitor SC79 (20 μΜ / L), the ability of PP VII (4 μM / L) to promote apoptosis can be reversed. When PP VII (4 μM / L) + CDDP (50 μΜ / L) are used together, the apoptosis of bladder cancer cells can be significantly promoted. After adding the apoptosis inhibitor SC79 (20 μΜ / L), the ability of PP VII (4 μM / L) + CDDP (50 μΜ / L) to promote apoptosis can be reversed.
[0022] (2) Western blot experiment Logarithmic growth phase T24CDDP cells were uniformly inoculated in a 6-well plate and placed in a cell culture box. After being cultured at 37°C and 5% CO2 for 24 hours, the cells were completely attached, and then treated with single drug PP VII (4 μM / L), CDDP (50 μΜ / L), and PP VII (4 μM / L) + CDDP (50 μΜ / L) for 24 hours. The total protein of the cells was extracted, and a western blot experiment was performed to detect the expression levels of c-PARP and c-cas-7 proteins. A blank control group without adding drugs was also set. The results are shown in Figure 6. As can be seen from the figure, PP VII (4 μM / L) and CDDP (50 μΜ / L) alone can promote the increase of the levels of c-PARP and c-cas-7 key apoptosis proteins. Compared with single drug administration, the levels of c-PARP and c-cas-7 key apoptosis proteins significantly increase when PP VII (4 μM / L) + CDDP (50 μΜ / L) are used together, indicating that PP VII (4 μM / L) + CDDP (50 μΜ / L) can further promote the apoptosis of human bladder cancer cells.
[0023] Example 3: Inhibition experiment of CDDP and PP VII on T24CDDP cells T24CDDP cells were evenly plated in 96-well plates (4000 cells / well, 100 μL per well), and incubated in a cell incubator at 37°C, 5% CO2 for 24 hours. After the cells were completely adherent, different concentrations (1 μM / L, 2 μM / L, 4 μM / L) of PPⅦ and cisplatin (CDDP) were combined and treated 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, 5% CO2 for 5 hours. The liquid in the plates was then discarded, 100 μL of DMSO solution (purity 99.99%) was added, and the plates were shaken for 10 minutes (200 rpm). The absorbance was measured at 570 nm, and the cell survival rate was calculated to draw the dose-effect curve. The results are shown in Figure 7 As can be seen from the figure, the proliferation of T24CDDP cells was inhibited under the action of cisplatin (CDDP) combined with PPⅦ, and PPⅦ inhibited the proliferation of cancer cells in a concentration-dependent manner. Compared with CDDP alone, PPⅦ enhanced the cytotoxicity of CDDP on T24CDDP cells.
[0024] Example 4: Evaluation of the in vivo anti-tumor effect of PPⅦ monotherapy and combination with cisplatin on T24CDDP transplanted tumors 1. Drug effect on mouse body weight and inhibition of tumor size BALB / c nu / nu male mice (13-17 g; 4-5 weeks) were purchased from the Animal Research and Resource Center of Yunnan University (Kunming, Yunnan).
[0025] The mice were raised at 25°C under a 12 / 12 hour light-dark cycle. After one week of adaptation to standard rodent feed, the mice were randomly divided into four groups: a blank control group, a CDDP group, a PPⅦ treatment group, and a CDDP+PPⅦ combination group. T24CDDP cells were resuspended in PBS solution containing 50% Corning Matrigel (Corning, New York, USA), and then injected subcutaneously into the right groin area of each mouse (3×10 6 cells per mouse). The long diameter (L; mm) and short diameter (W; mm) of the xenotransplanted tumors were measured regularly, and the tumor volume (V; mm 3 ) was calculated according to the formula (V=0.5×L×W 2 ). When the transplanted tumor volume reached 50-100 mm 3 , the mice were injected intraperitoneally with drug (PBS solution containing 50% Corning Matrigel, blank control, once every 2 days), CDDP (2 mg / kg, once every 2 days), PPⅦ (2 mg / kg, once every 2 days), and CDDP+PPⅦ treatment. On day 14, the body weight of the mice was measured. The mice were sacrificed (carbon dioxide anesthesia), dissected, and the tumors were removed, measured for volume, and weighed.
[0026] The results are shown in Figures 8-9. Animal experiments show that Figure 8 It can be seen that in the T24CDDP transplant tumor model, PPVII monotherapy showed significant tumor growth inhibition, and when combined with cisplatin, it produced a significant synergistic anti-tumor effect, with tumor volume and weight significantly reduced, and the drug interaction coefficient was 0.45. Figure 9 It can be seen that PPVII alone does not cause significant weight changes in experimental animals, and when used in combination with cisplatin, it does not aggravate the weight loss caused by cisplatin.
[0027] 2. H&E staining experiment of mouse tumor tissue Samples were fixed with 4% paraformaldehyde at 4°C for 24 hours and then dehydrated with 70%, 80%, 90%, 95%, and 100% ethanol for 1 hour each. They were cleared twice with xylene for 30 minutes each. Finally, they were embedded in paraffin at 60°C. After embedding, 4 μm sections were cut and baked at 60°C for 2 hours. Prior to staining, the sections were deparaffinized with xylene and ethanol (100%, 95%, and 80%), stained with hematoxylin and eosin, and then dehydrated and mounted.
[0028] The results are shown in Figure 10. Compared with the blank control group, the CDDP monotherapy group demonstrated vacuolation and nuclear pyknosis in some cells, with lesions present in approximately 30% of the cells. The PPVII monotherapy group showed prominent apoptotic bodies, indicating widespread apoptosis, with over 60% of cells exhibiting nuclear fragmentation. The combination therapy group demonstrated significant inflammatory cell infiltration, extensive cellular necrosis, and only a small number of surviving tumor cells. Overall, H&E staining results demonstrated that PPVII monotherapy significantly inhibited tumor growth in the T24CDDP xenograft model, while its combination with cisplatin produced a significant synergistic anti-tumor effect.
Claims
1. Application of Paris polyphylla saponin VII in the preparation of chemotherapy sensitizer for bladder cancer.
2. Application of Paris polyphylla saponin VII in combination with cisplatin in the preparation of drugs for the treatment of bladder cancer.
3. The use according to claim 1, characterized in that: Paris polyphylla saponin VII overcomes cisplatin resistance by inhibiting the Akt-SREBP1 lipid metabolism pathway.