Application of isoalantolactone in preparation of medicine for treating liver cancer

Abstract

The invention discloses an application of isoalantolactone in preparation of a medicine for treating liver cancer, and through systematic in-vivo and in-vitro experimental research, the action effect of the isoalantolactone on normal liver cells of people and three liver cancer cell lines is evaluated. Various technical methods such as cell viability detection, phenotype analysis, marker protein detection, target verification, nude mouse ectopic transplantation tumor model and the like are adopted, and it is proved that the isoalantolactone plays a role in resisting liver cancer through specific targeting PCSK9. The method has the characteristics of simplicity and convenience in operation, good repeatability, remarkable effect and the like. The research discloses the application potential of the isoalantolactone as the PCSK9 targeted inhibitor in anti-liver cancer treatment for the first time, an important lead compound is provided for developing a novel PCSK9 targeted anti-liver cancer drug, and the discovery provides a new research direction and treatment strategy for targeted treatment of liver cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmacological evaluation of monomeric compounds, specifically to the application of isothomyl lactone in the preparation of a drug for treating liver cancer. 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 manifested as 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 development directions for innovative drug research in my country. *Inula japonica*, belonging to the genus *Inula* of the Asteraceae family, is bitter, pungent, and warm in nature. Its roots can be used medicinally, possessing the effects of harmonizing the stomach and spleen, regulating qi, and relieving depression. Isoalantolactone (IATL) is a sesquiterpene lactone small molecule compound extracted from *Inula japonica*, possessing various medicinal values ​​such as anti-inflammatory, antibacterial, and anticancer properties. Isoalantolactone has been proven to inhibit the proliferation and induce apoptosis in various tumor cell lines such as lung cancer, gastric cancer, and prostate cancer. However, research on isoalantolactone in the treatment of liver cancer is limited, and the related mechanisms of action are not systematically studied. Therefore, this invention aims to explore the in vitro and in vivo pharmacodynamics of isoalantolactone against human liver cancer cells and to investigate its anticancer mechanism. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide the application of isothomolecin lactone in the preparation of a drug for treating liver cancer; another objective of the present invention is to provide a pharmaceutical composition for treating liver cancer. The present invention elucidates the in vitro and in vivo anti-liver cancer therapeutic effects of isothomolecin lactone, explores the anti-liver cancer mechanism of isothomolecin lactone targeting PCSK9, provides a theoretical basis and lead compounds for the development of PCSK9-targeting inhibitors, and offers new ideas for liver cancer treatment.

[0005] To achieve the above objectives, the present invention provides the following technical solution: 1. Use of isoalantolactone in the preparation of a medicament for treating liver cancer, wherein the isoalantolactone exerts an anti-liver cancer effect by targeting proprotein convertase subtilisin 9.

[0006] In some embodiments of the present application, the use comprises administering isoalantolactone to an individual in need of treatment to inhibit liver cancer cell proliferation and induce liver cancer cell apoptosis.

[0007] In some embodiments of the present application, the isoalantolactone exerts an anti-liver cancer effect by at least one of the following mechanisms: a) increasing the level of reactive oxygen species in liver cancer cells; b) reducing the mitochondrial membrane potential of liver cancer cells; c) inducing mitochondrial morphological fragmentation and fusion; d) up-regulating the expression of pro-apoptotic proteins Bax and Cleaved caspase-3 and down-regulating the expression of anti-apoptotic protein Bcl-2.

[0008] In some embodiments of the present application, the isoalantolactone directly binds to the proprotein convertase subtilisin 9 protein, and the binding is verified by at least one of the following methods: drug affinity reaction targeting stability experiment, solvent-induced protein precipitation experiment, or small molecule Pull-down experiment.

[0009] In some embodiments of the present application, the medicament is administered by in vivo administration, and the administration dose is 20 mg / Kg to 40 mg / Kg, with once-a-day administration every other day.

[0010] In some embodiments of the present application, the isoalantolactone exerts an anti-liver cancer effect in vivo by reducing the expression level of proprotein convertase subtilisin 9 in tumor tissue.

[0011] In some embodiments of the present application, the treatment of isoalantolactone can cause cell death and reduced proliferation ability in tumor tissue, and has no significant toxic effect on the liver and kidney functions of nude mice, showing good safety.

[0012] 2. A pharmaceutical composition for treating liver cancer, comprising a therapeutically effective amount of isoalantolactone and a pharmaceutically acceptable carrier or excipient.

[0013] The beneficial effects of this invention are as follows: This invention provides a small-molecule anti-hepatocellular carcinoma inhibitor that can target PCSK9. This invention, by investigating the effects of isothomyl lactone on the survival of liver cancer cells, changes in mitochondrial function, apoptosis levels, target binding, and in vivo anti-hepatocellular carcinoma activity, provides a new lead compound and potential therapeutic target for liver cancer treatment. This method is convenient, rapid, simple to operate, has significant effects, and good reproducibility. This invention discovers and confirms that isothomyl lactone treatment exhibits good anti-hepatocellular carcinoma effects in vitro and in vivo. This invention can provide theoretical guidance for the comprehensive development and utilization of isothomyl lactone and provide potential lead compounds and targets for liver cancer treatment. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 The study showed the effects of different doses of isothomyl lactone intervention on the viability and migration ability of normal liver cells (MIHA) and liver tumor cells (Huh-7, Hep3B, HepG2) (ns means no significant, *p<0.05, **p<0.01, ***p <0.001). Figure 2 This study demonstrated the effects of different doses of isothomyl lactone intervention on mitochondrial-related functions in hepatocellular carcinoma cells. Figure 3 This study demonstrated the effects of different doses of isothomyl lactone intervention on the apoptosis level of liver cancer cells. Figure 4 This indicates that isothomyl lactone has a stable binding with PCSK9; Figure 5 The study showed that isothomyl lactone inhibited the growth of Huh-7 xenografts in nude mice (**p<0.01,***p<0.001). Figure 6 The effects of isothomyl lactone on cell death and proliferation in tumor tissue cells were shown. Figure 7 This study demonstrated the effect of isothomyl lactone on the expression level of PCSK9 in tumor tissue cells; Figure 8 The study showed the effect of isothomyl lactone on the safety of nude mice (ns means no significant). Detailed Implementation

[0015] 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.

[0016] Example 1, direct action of isoalantolactone on cells 1. Dose and time of administration Human normal liver cells MIHA and liver cancer cells, including Huh-7, Hep3B and HepG2, were selected as cell lines, the culture medium was DMEM and MEM-α, the drug concentration was 2.5, 5.0, 10.0 μM, and the drug administration time was 48 h. As shown in Figure 1 , the proliferation of liver cancer cells was inhibited under the intervention of different concentrations of isoalantolactone.

[0017] 2. Detection of cellular reactive oxygen species (ROS) The cells were detected by ROS kit. As shown in Figure 2 , A, under the intervention of different concentrations of compounds, the release of reactive oxygen species showed an upward trend and was dose-dependent. ROS can be used as an early marker of apoptosis, indicating that isoalantolactone may induce apoptosis of liver cancer cells.

[0018] 3. Detection of mitochondrial membrane potential (JC-1) of cells The cells were detected by JC-1 nucleic acid dye. As shown in Figure 2 , B, under the intervention of different concentrations of compounds, the red fluorescence gradually weakened and the green fluorescence gradually increased, indicating that the mitochondrial membrane potential in cells decreased and was dose-dependent, indicating that isoalantolactone caused damage to liver cancer cells.

[0019] 4. Detection of mitochondrial morphology of cells Tumor cells were detected by Mito-tracker green fluorescent dye. As shown in Figure 2 , C, under the intervention of 10.0 μM isoalantolactone, the mitochondrial morphology was broken and fused.

[0020] 5. Detection of cell apoptosis The cells were detected by cell cycle and apoptosis detection kit. As shown in Figure 3 , A, under the intervention of different concentrations of isoalantolactone, the apoptosis level showed a dose-dependent increase, indicating that isoalantolactone had cytotoxicity and could induce apoptosis of liver cancer cells.

[0021] 6. Detection of apoptosis protein level of cells The protein expression level was detected by Western blotting. As shown in Figure 3 , B-D, isoalantolactone could up-regulate the expression of pro-apoptotic proteins Bax and Cleaved caspase-3 proteins in cells, while down-regulate the expression level of anti-apoptotic protein Bcl-2, and showed a dose-dependent manner, indicating that isoalantolactone induced apoptosis of cells.

[0022] Example 2, Target binding experiment of isoalantolactone and PCSK9 1, Drug affinity reaction target stability detection experiment The concentration of isoalantolactone is 0, 2.5, 5.0 and 10.0 μM, and the protease acts on tumor cells for 8 minutes. The protein expression level of PCSK9 (proprotein convertase subtilisin 9) is detected. As shown in Figure 4 , A, short-term intervention of isoalantolactone restores the decrease of PCSK9 protein expression level caused by protease.

[0023] 2, Solvent-induced protein precipitation detection experiment After 10.0 μM of isoalantolactone is co-incubated with liver cancer cell lysate, acetone-absolute ethanol-acetic acid mixture (AEA) is added in the control group and the drug group, respectively, and the binding degree of isoalantolactone and PCSK9 is detected by immunoblotting technology at different temperatures. As shown in Figure 4 , B, the intervention of isoalantolactone improves the expression level of PCSK9 protein, indicating that isoalantolactone and PCSK9 exist binding.

[0024] 3, Small molecule pull-down experiment After the biotin-attached isoalantolactone is co-incubated with Huh-7 cell lysate, streptavidin is added, and the mutual binding of isoalantolactone and PCSK9 is detected by immunoblotting technology. As shown in Figure 4 , C, biotin-attached isoalantolactone can bind to PCSK9.

[0025] Example 3, In vivo anti-tumor experiment of isoalantolactone 1, Establishment and treatment of nude mouse ectopic tumor model The well-conditioned Huh-7 cells are digested, centrifuged, and injected with 5×10 6 cells per 100 microliters of PBS. Each nude mouse is injected with 100 microliters of tumor cell suspension. After the tumor is formed, it is randomly divided into a control group, a low-dose drug group and a high-dose drug group, and treated with isoalantolactone (20 mg / Kg and 40 mg / Kg). The drug is given every other day, and the changes in tumor volume and body weight of nude mice are recorded. As shown in Figure 5 , A-C, 20 mg / Kg and 40 mg / Kg of isoalantolactone significantly inhibit the growth of liver tumors and reduce the weight of liver tumors. As shown in Figure 5 , D, isoalantolactone does not significantly affect the body weight of nude mice.

[0026] 2, Hematoxylin-eosin staining experiment The paraffin section was dewaxed, stained with hematoxylin for 3 minutes, and washed with distilled water to remove the floating color. The tissue was differentiated in 1% hydrochloric acid alcohol differentiation solution for 30 seconds and then washed with tap water. The tissue was stained with eosin, and after staining for 2 minutes, it was washed with distilled water. The tissue was gradient dehydrated and mounted. As shown in Figure 6 , A, 20 mg / Kg and 40 mg / Kg of isoalantolactone treatment caused the tumor tissue inside the nude mice to be loose, and the nucleocytoplasmic separation phenomenon appeared. As shown in Figure 6 , B, 20 mg / Kg and 40 mg / Kg of isoalantolactone treatment caused the distribution of tumor proliferation marker Ki-67 to decrease. As shown in Figure 6 , C, 20 mg / Kg and 40 mg / Kg of isoalantolactone treatment showed an increasing trend in the apoptosis level in tumor tissue.

[0027] 3. Immunohistochemical staining experiment The paraffin section was dewaxed, and the tissue was subjected to microwave antigen repair and then naturally cooled. Triton X-100 was added dropwise on the tissue to permeabilize the cells, and the tissue was soaked in 3% hydrogen peroxide solution for 30 minutes. After the tissue section was blocked with serum for 30 minutes, the first antibody was added dropwise and incubated at 4°C overnight. The tissue section was continuously added with specific secondary antibody and incubated for 1.5 hours. After the tissue section was washed with PBS, DAB color development was performed, hematoxylin was added to stain the nucleus, and then the tissue was mounted. As shown in Figure 7 , A and B, after 20 mg / Kg and 40 mg / Kg of isoalantolactone treatment, the distribution and protein expression level of PCSK9 in liver tumor tissue decreased.

[0028] 4. Safety experiment of isoalantolactone on nude mice Alanine / aminotransferase detection and creatinine detection. The nude mice were sacrificed according to the ethical requirements, and the mouse serum was collected. Alanine aminotransferase kit and creatinine detection kit were used and the detection was performed according to the instructions in the kit. As shown in Figure 8 , after 20 mg / Kg and 40 mg / Kg of isoalantolactone treatment, no hepatotoxicity and nephrotoxicity was caused in nude mice.

[0029] The above examples are only preferred examples for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. Use of isoalantolactone for the preparation of a medicament for the treatment of liver cancer, characterized in that, The isodehydroalantolactone exerts an anti-hepatoma effect by targeting proprotein convertase subtilisin 9.

2. Use according to claim 1, characterized in that, The application comprises administering isodehydroalantolactone to an individual in need of treatment to inhibit hepatoma cell proliferation and induce hepatoma cell apoptosis.

3. Use according to claim 1 or 2, characterized in that, The isodehydroalantolactone exerts an anti-hepatoma effect by at least one of the following mechanisms: a) increasing the level of reactive oxygen species in hepatoma cells; b) reducing the mitochondrial membrane potential of hepatoma cells; c) inducing mitochondrial morphological disruption and fusion; d) up-regulating the expression of pro-apoptotic proteins Bax and Cleaved caspase-3 and down-regulating the expression of anti-apoptotic protein Bcl-2.

4. Use according to claim 1, characterized in that, The isodehydroalantolactone directly binds to proprotein convertase subtilisin 9 protein, and the binding is verified by at least one of the following methods: drug affinity reaction targeting stability experiment, solvent-induced protein precipitation experiment, or small molecule Pull-down experiment.

5. The use according to claim 1, characterized in that, The drug is administered by in vivo administration, and the administration dose is 20 mg / Kg to 40 mg / Kg, and the administration is once every other day.

6. Use according to claim 5, characterized in that, The isodehydroalantolactone exerts an anti-hepatoma effect in vivo by reducing the expression level of proprotein convertase subtilisin 9 in tumor tissues.

7. Use according to claim 5, characterized in that, The treatment of isodehydroalantolactone can cause cell death and reduced proliferation ability of tumor tissues, and has no significant toxic effect on the liver and kidney functions of nude mice, and shows good safety.

8. A pharmaceutical composition for treating liver cancer, characterized by, The isodehydroalantolactone comprises a therapeutically effective amount of isodehydroalantolactone and a pharmaceutically acceptable carrier or excipient.

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