Use of schisantherin a in preparation of liver cancer cell aging inducer and combination scheme with sorafenib

Abstract

The application discloses application of schisantherin A in preparation of a liver cancer cell aging inducer and a combined use scheme of sorafenib, discloses feasibility of Sirt6 as a new target of anti-liver cancer drugs for the first time, and finds that schisantherin A is a small molecule lead compound capable of efficiently and targetingly combining with Sirt6. The application fully confirms the anti-liver cancer effect of schisantherin A in targeting Sirt6 from three levels of molecule, cell and whole animal by systematically investigating the influence of schisantherin A and the combination of schisantherin A and sorafenib on liver cancer cell survival rate, proliferation activity and aging phenotype, combining with in-vitro verification of target binding mechanics and in-vivo anti-liver cancer pharmacodynamics evaluation, improves the clearance efficiency of sorafenib on aging liver cancer cells, effectively avoids the problem of non-specific cytotoxicity in traditional combined use of drugs, and provides a new idea for precise and sequential treatment of liver cancer.

Description

Technical Field

[0001] This invention relates to the field of medicinal plant extracts, specifically to the application of schisandrin A in the preparation of senescence-inducing agents for liver cancer cells, and also to the application of a combination of schisandrin A and sorafenib in the preparation of anti-liver cancer drugs. Background Technology

[0002] Liver cancer is a highly heterogeneous malignant tumor, and its occurrence and development are closely related to metabolic disorders. Statistics show that approximately 410,000 new cases of liver cancer are diagnosed in my country each year, accounting for about 45% of new cases globally. Its mortality rate ranks second among all malignant tumors in my country, seriously threatening human life and health. Currently, sorafenib is widely used as a first-line drug for the clinical treatment of liver cancer. However, this drug has significant toxic side effects during use, mainly skin toxicity and hematologic toxicity, which seriously affect treatment efficacy and even lead to treatment interruption. Therefore, the search for safe and effective new anti-liver cancer drugs is extremely urgent.

[0003] Traditional Chinese medicine (TCM), as a unique health resource and a potentially huge economic resource in my country, plays an important role in the overall economic and social development of the country and is receiving increasing attention worldwide. In the field of new drug development, TCM has become a source of innovative drugs and one of the important directions for innovative drug development in my country. Schisandra chinensis is a traditional tonic TCM herb with five flavors: sweet, sour, bitter, pungent, and salty. It has the effects of astringing the lungs and kidneys, promoting body fluid production and reducing sweating, astringing essence and stopping diarrhea, and calming the mind and soothing the nerves. Clinically, it is used for symptoms such as chronic cough and asthma, nocturnal emission, persistent diarrhea, night sweats, palpitations, and insomnia. Modern pharmacological studies have also found that Schisandra chinensis has good anti-tumor activity, inhibiting tumor cell proliferation by inducing apoptosis and regulating the tumor microenvironment (inhibiting angiogenesis and inflammatory responses, etc.) to achieve anti-tumor effects. Schisandra chinensis ester A is a biphenylcyclooctadiene lignan compound extracted from Schisandra chinensis, possessing multiple medicinal values ​​such as hepatoprotection, anti-inflammation, and anti-cancer properties. Schisandra chinensis has been shown to inhibit the proliferation and induce apoptosis in various tumor cell lines, such as liver cancer, pancreatic cancer, breast cancer, and prostate cancer. However, research on the treatment of liver cancer with schisantherin A (SchA) is limited, and the related mechanism of action is not systematically studied. Therefore, this invention aims to explore the in vivo and in vitro pharmacodynamics of schisantherin A against human liver cancer cells and its mechanism of action, and to investigate its in vivo and in vitro pharmacodynamics in combination with the first-line anti-liver cancer drug sorafenib. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide the application of schisandrin A in the preparation of a liver cancer cell senescence inducer; a second objective of the present invention is to provide the application of schisandrin A in the preparation of a Sirt6 targeting inhibitor; a third objective of the present invention is to provide the application of schisandrin A in the preparation of an anti-liver cancer drug; and a fourth objective of the present invention is to provide the application of a combination of schisandrin A and sorafenib in the preparation of an anti-liver cancer drug.

[0005] To achieve the above objectives, the present invention provides the following technical solution: Application of Schisandrin A in the preparation of senescence inducers for liver cancer cells.

[0006] Application of Schisandrin A in the preparation of Sirt6-targeting inhibitors.

[0007] Schisandrin A is used in the preparation of anti-hepatocellular carcinoma drugs. Schisandrin A exerts its anti-hepatocellular carcinoma effect by targeting and inhibiting Sirt6 protein expression, thereby inducing senescence phenotype in liver cancer cells.

[0008] Application of the combination of schisandrin A and sorafenib in the preparation of anti-hepatocellular carcinoma drugs.

[0009] Preferably, the mass ratio of schisandrin A to sorafenib in this invention is 10:1.

[0010] In this invention, the preferred dosage of schisandrin A is 100 mg / kg, and the preferred dosage of sorafenib is 10 mg / kg.

[0011] The pharmaceutical composition exerts its anti-hepatocellular carcinoma effect through the following mechanism: Schisandra chinensis ester A induces hepatocellular carcinoma cells to enter a senescent state, increasing the senescence-associated vulnerability phenotype, which is then selectively cleared by sorafenib.

[0012] The beneficial effects of this invention are as follows: This invention reveals for the first time the feasibility of Sirt6 as a novel target for anti-hepatocellular carcinoma drugs, and discovers that schisandrin A is a small molecule lead compound that can efficiently target and bind Sirt6. This invention systematically investigates the effects of schisandrin A on the survival rate, proliferation activity, and aging phenotype of hepatocellular carcinoma cells, combined with in vitro validation of target binding mechanics and in vivo pharmacodynamic evaluation of its anti-hepatocellular carcinoma effects, fully demonstrating its anti-hepatocellular carcinoma targeting effect on Sirt6 at the molecular, cellular, and whole animal levels. This provides a candidate lead compound and a new intervention target with independent intellectual property rights for the treatment of hepatocellular carcinoma.

[0013] This invention also proposes and validates a novel staged combination drug therapy for liver cancer based on a "first induce senescence, then target and clear" strategy, namely, the combination of schisandrin A and sorafenib. Its mechanism of action is as follows: schisandrin A preferentially acts on liver cancer cells, inducing them into an irreversible senescent state, thereby significantly increasing the senescence-associated vulnerability phenotype; based on this, sorafenib achieves selective and efficient clearance of senescent liver cancer cells. This treatment strategy not only improves the clearance efficiency of sorafenib against senescent liver cancer cells but also effectively avoids the non-specific cytotoxicity problem in traditional combination therapies, providing a new approach for the precision and sequential treatment of liver cancer.

[0014] The combined dosing regimen established in this invention has outstanding advantages such as convenient operation, controllable timing, significant efficacy, and good batch-to-batch reproducibility, demonstrating good feasibility for clinical translation and promising application prospects. This invention uncovers new anti-tumor applications from the classic Chinese herbal active ingredient, Schisandra chinensis ester A, not only expanding the depth and breadth of its comprehensive development and utilization, but also providing strong theoretical basis and experimental support for the discovery of lead compounds for liver cancer treatment and the verification of new targets. Attached Figure Description

[0015] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 The effects of different doses of schisandrin A on the viability and proliferation of liver tumor cells; Figure 2 The effect of different doses of schisandrin A intervention on the senescence level of liver cancer cells; Figure 3 Schisandrin A and Sirt-6 are stably bound together; Figure 4 The inhibitory effect of schisandrin A on Huh-7 heterotopic xenografts in nude mice; Figure 5 The effect of schisandrin A combined with sorafenib on the proliferation of liver cancer cells; Figure 6 The effect of schisandrin A combined with sorafenib on the senescence level of liver cancer cells; Figure 7 The study investigated the inhibitory effect of schisandrin A combined with sorafenib on Huh-7 heterotopic xenografts in nude mice. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0017] Example 1: Detection of relevant indicators after schisandrin A was directly applied to human hepatocellular carcinoma cell lines Huh7 and HepG2. 1. Dosage and timing of administration Human liver cancer cells Huh7 and HepG2 were selected, and the culture media were DMEM and MEM-α, respectively. The concentrations of schisandrin A were 0, 25.0, 50.0 and 100.0 μM, and the administration time was 7 days.

[0018] 2. MTT assay The survival rate of liver cancer cells was detected by the MTT assay, and the results were as follows: Figure 1 As shown in Figure A, under different concentrations of schisandrin A, the survival rate of liver cancer cells showed a downward trend and exhibited a dose-dependent effect.

[0019] 3. Colony cloning detection Hepatocellular carcinoma cells were analyzed for proliferation using a colony-cloning method. Results are as follows: Figure 1 As shown in Figures B and C, the number of cell colonies decreased under different concentrations of schisandrin A, and this decrease was dose-dependent, indicating that schisandrin A has an inhibitory effect on the proliferation of tumor cells.

[0020] 4. Long-term proliferation level detection Long-term proliferation experiments were used to detect the proliferative capacity of liver cancer cells. Results are as follows: Figure 1 As shown in Figure D, under different concentrations of schisandrin A, the proliferation generation of liver cancer cells was significantly reduced, indicating that schisandrin A has an inhibitory effect on the proliferation of liver cancer cells.

[0021] Example 2: Detection of relevant indicators of schisandrin A directly promoting the senescence of liver cancer cells. 1. Detection of cellular β-galactosidase staining The detection was performed using a cellular β-galactosidase staining kit. Results are as follows: Figure 2 As shown in A and B, under different concentrations of schisandrin A intervention, the level of cell senescence increased in a dose-dependent manner, indicating that schisandrin A induces senescence in liver cancer cells.

[0022] 2. Detection of Reactive Oxygen Species (ROS) in Cells The reactive oxygen species (ROS) levels in liver cancer cells were detected using a ROS detection kit. Results are as follows: Figure 2 As shown in C and D, schisandrin A can induce ROS accumulation in liver cancer cells in a dose-dependent manner, indicating that schisandrin A induces a senescent phenotype in liver cancer cells.

[0023] 3. Detection of cell senescence-related proteins Western blot was used to detect aging-related proteins in liver cancer cells. Results are as follows: Figure 2As shown in E and F, schisandrin A can cause a dose-dependent decrease in Lamin B1 protein expression while activating p53 and p21 protein expression, which together indicate that schisandrin A leads to hepatocyte senescence.

[0024] Example 3: Detection of relevant indicators of schisandrin A targeting and inhibiting Sirt-6 protein expression 1. Micro-heat surge experiment Sirtuin 6 (Sirt6) protein and schisandrin A were tested using a micro-thermal surge experiment conducted by a specialized biotechnology company. The results are as follows: Figure 3 As shown in Figure A, schisandrin A can bind to Sirt6 with moderate strength, with a Kd value of 3.55 ± 0.32 μM.

[0025] 2. Drug affinity response and target stability assay Schisandra chinensis ester A at concentrations of 0, 25.0, 50.0, and 100.0 μM was applied to tumor cells with the intervention of thermophilic bacteria protein, and the protein expression level of Sirt6 was detected. The results are as follows: Figure 3 As shown in Figure B, intervention with schisandrin A increased Sirt6 protein expression levels.

[0026] 3. Solvent-induced protein precipitation detection experiment After co-incubating 100.0 μM schisandrin A with liver cancer cell lysis buffer, an acetone-anhydrous ethanol-acetic acid mixture was added to both the control and treatment groups. The binding degree of schisandrin A to Sirt6 was detected using Western blotting at different temperatures. Results are as follows: Figure 3 As shown in Figure C, schisandrin A increased the expression level of Sirt6 protein, indicating that schisandrin A binds to Sirt6.

[0027] Example 4: Detection of related indicators after Schisandra chinensis ester A targeted inhibition of Sirt6 protein expression and inhibition of liver cancer cell proliferation in mice. 1. Establishment and treatment of nude mouse ectopic xenograft models Healthy Huh7 cells were digested, centrifuged, and diluted with 5 × 10⁵ cells per 100 μL PBS. 6 Cell injection was performed on each nude mouse. Each mouse was injected with 100 μL of tumor cell suspension. After tumor formation, the mice were randomly divided into a control group, a low-dose treatment group, and a high-dose treatment group, and treated with schisandrin A (50.0 mg / Kg and 100.0 mg / Kg) every other day. Tumor volume and body weight changes were recorded. Results are as follows: Figure 4 As shown in A to D, schisandrin A treatment significantly inhibited the proliferation of liver cancer cells.

[0028] 2. Hematoxylin-eosin staining experiment Paraffin sections were dewaxed, stained with hematoxylin for 3 minutes, and washed with distilled water to remove excess stain. The tissues were then placed in 1% hydrochloric acid-alcohol differentiation medium for 30 seconds, followed by rinsing with tap water. Eosin staining was performed on the tissues for 2 minutes, followed by rinsing with distilled water. The tissues were then dehydrated in a gradient and mounted for observation. Results are as follows: Figure 4 As shown in E, treatment with schisandrin A induced nucleocytoplasmic separation in mouse liver cancer tissue.

[0029] 3. Immunohistochemical staining experiment Paraffin sections were dewaxed, and after microwave antigen retrieval, the tissues were allowed to cool naturally. Triton X-100 was added to the tissues for cell permeation, and the sections were then immersed in 3% hydrogen peroxide solution for 30 minutes. The tissue sections were blocked with serum for 30 minutes, then primary antibody was added, and incubated overnight at 4 °C. Specific secondary antibody was added to the tissue sections, and incubation continued for 1.5 hours. After washing the tissue sections with PBS, DAB staining was performed, and hematoxylin was added to stain the nuclei before mounting and observation. Results are as follows: Figure 4 As shown in F and G, treatment with schisandrin A significantly reduced the Ki-67 content in mouse liver cancer tissue.

[0030] Example 5: In vivo and in vitro detection of relevant indicators after the combined use of schisandrin A and sorafenib. The relevant experimental techniques and operations are as described in Examples 1 (2 and 3), Examples 2 (1 and 3), and Examples 4 (1-3).

[0031] 1. Dosage and timing of administration Human liver cancer cells Huh7 and HepG2 were selected, and the culture media were DMEM and MEM-α, respectively. The concentration of schisandrin A was 100.0 μM, and the concentration of sorafenib was 10.0 μM. The administration time was 2 days.

[0032] 2. MTT assay The survival rate of liver cancer cells was detected by the MTT assay, and the results were as follows: Figure 5 As shown in Figure A, the survival rate of liver cancer cells showed a downward trend under the combined intervention of schisandrin A and sorafenib, and this trend was dose-dependent.

[0033] 3. Colony cloning detection Hepatocellular carcinoma cells were analyzed for proliferation using a colony-cloning method. Results are as follows: Figure 5 As shown in Figures B and C, the number of cell colonies was significantly reduced under the combined intervention of schisandrin A and sorafenib compared to the treatment of schisandrin A and sorafenib alone, indicating that the combined intervention of schisandrin A and sorafenib has an inhibitory effect on the proliferation of tumor cells.

[0034] 4. Detection was performed using a cellular β-galactosidase staining kit. Results are as follows: Figure 6 As shown in A and B, the level of cellular senescence was not significantly increased under the combined intervention of schisandrin A and sorafenib compared with schisandrin A alone, indicating that the combined use of schisandrin A and sorafenib did not further trigger the senescence phenotype.

[0035] 5. Senescence-related proteins in liver cancer cells were detected using Western blot technology. Results are as follows: Figure 6 As shown in Figure C, the combination of schisandrin A and sorafenib did not further activate the expression of p53 and p21 proteins or reduce the expression of Lamin B1 protein compared with schisandrin A alone. This suggests that the anti-tumor effect of schisandrin A and sorafenib does not depend on cell senescence. Instead, schisandrin A first causes cell senescence, which is then further eliminated by sorafenib.

[0036] 6. Establishment and treatment of nude mouse ectopic xenograft models Healthy Huh7 cells were digested, centrifuged, and diluted with 5 × 10⁵ cells per 100 μL PBS. 6 Cell injection was performed on each nude mouse. Each mouse was injected with 100 μL of tumor cell suspension. Once tumors formed, mice were randomly divided into a control group, a schisandrin A treatment group, a sorafenib treatment group, and a combined schisandrin A and sorafenib treatment group. Treatment with schisandrin A and sorafenib, as well as the combined treatment, was administered at concentrations of (100.0 mg / kg, 10 mg / kg, and 100.0 mg / kg + 10 mg / kg), every other day. Tumor volume and body weight changes were recorded. Results are as follows: Figure 7 As shown in A to D, the combination of schisandrin A and sorafenib significantly inhibited the proliferation of liver cancer cells.

[0037] 7. Hematoxylin-eosin staining experiment Paraffin sections were dewaxed, stained with hematoxylin for 3 minutes, and washed with distilled water to remove excess stain. The tissues were then placed in 1% hydrochloric acid-alcohol differentiation medium for 30 seconds, followed by rinsing with tap water. Eosin staining was performed on the tissues for 2 minutes, followed by rinsing with distilled water. The tissues were then dehydrated in a gradient and mounted for observation. Results are as follows: Figure 7 As shown in E, the combination of schisandrin A and sorafenib caused the most severe nucleocytoplasmic segregation in mouse liver cancer tissue.

[0038] 8. Immunohistochemical staining experiment Paraffin sections were dewaxed, and after microwave antigen retrieval, the tissues were allowed to cool naturally. Triton X-100 was added to the tissues for cell permeation, and the sections were then immersed in 3% hydrogen peroxide solution for 30 minutes. The tissue sections were blocked with serum for 30 minutes, then primary antibody was added, and incubated overnight at 4 °C. Specific secondary antibody was added to the tissue sections, and incubation continued for 1.5 hours. After washing the tissue sections with PBS, DAB staining was performed, and hematoxylin was added to stain the nuclei before mounting and observation. Results are as follows: Figure 7 As shown in F and G, the combination of schisandrin A and sorafenib showed the strongest inhibitory effect on Ki-67 content in mouse liver cancer tissue.

[0039] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. Application of Schisandrin A in the preparation of senescence inducers for liver cancer cells.

2. Application of Schisandrin A in the preparation of Sirt6-targeting inhibitors.

3. The application of schisandrin A in the preparation of anti-hepatocellular carcinoma drugs, characterized in that: The schisandrin A exerts its anti-liver cancer effect by targeting and inhibiting Sirt6 protein expression, thereby inducing a senescent phenotype in liver cancer cells.

4. Application of the combination of schisandrin A and sorafenib in the preparation of anti-hepatocellular carcinoma drugs.

5. The application according to claim 4, characterized in that: The mass ratio of schisandrin A to sorafenib is 10:

1.

6. The application according to claim 4, characterized in that: The dosage of schisandrin A is 100 mg / kg, and the dosage of sorafenib is 10 mg / kg.

7. The application according to claim 4, characterized in that: The pharmaceutical composition exerts its anti-hepatocellular carcinoma effect through the following mechanism: Schisandra chinensis ester A induces hepatocellular carcinoma cells to enter a senescent state, increasing the senescence-associated vulnerability phenotype, which is then selectively cleared by sorafenib.

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