Anti-cancer Activity And Its Mechanism Of Glycyrrhetinic Acid Methyl Ester Derivative PG-4C Containing Piperazine Structure

BackgroundGallbladder carcinoma(GBC)is a common tumor of the biliary system,which is highly invasive,malignant and fatal.Breast cancer(BRC)is a common malignant tumor originating from breast epithelial cells,which ranks the first in the incidence of female cancer,accounting for up to 24.2%.Peroxisome proliferator-activated receptor γ(PPARy)is a member of the nuclear receptor superfamily,it is involved in a variety of physiological and pathological processes.Glycyrrhiza is one of the commonly used Chinese herbal medicines in clinical practice.It has the functions of harmonizing various drugs and alleviating drug toxicity.Glycyrrhetinic acid is the main active component of glycyrrhiza,which is often used in combination with chemotherapy drugs to alleviate the side effects of drugs.Piperazine,also known as hexahydropyrazine,is an important cyclic amine compound that is often used to make synthetic antibiotics such as quinolones.In this thesis,we tested the activity and mechanism of a piperazin-containing methyl glycyrrhetinic acid derivative(PG-4C)against gallbladder and breast cancer,hoping to provide a new therapeutic strategy for these cancers by exploring a novel,effective and low-toxic chemotherapy drug combined with immunotherapy.Thus increasing the survival time of these patients and improving their prognosis.Objective1.Using PPARy protein as drug target,the feasibility of PG-4C as a PPARy agonist was evaluated by molecular docking technology.2.To evaluate the pharmacological activity of PG-4C,a novel PPARγ agonist,against gallbladder and breast cancer.3.To analyze the anti-gallbladder and anti-breast cancer mechanism of PG-4C.Methods1.Molecular docking experiments.Discovery Studio was used to analyze the molecular docking model of target compound PG-4C and PPARγ 3D X-ray structure through the graphical user interface DS-CDOCKER protocol.2.In vitro experiments.Including cell morphology observation;In vitro anti-tumor activity assay;Cell cycle analysis;Apoptosis analysis experiment;Mitochondrial membrane potential measurement experiment;Intracellular Ca2+measurement experiment;Scratch test;Cell migration and invasion assay;Immunofluorescence staining,confocal microscopy analysis and Western Blot analysis of actin filaments were performed.3.In vivo experiments.This included evaluation of the inhibitory effect of PG-4C on the growth of xenograft gallbladder cancer in zebrafish and xenograft breast cancer in mice.4.Drug toxicity of PG-4C was evaluated.Wild and transgenic zebrafish embryos(labeled with vascular or hematopoietic stem cells)at 48hpf were treated with a medicinal bath with the target concentration of PG-4C and positive controls pioglitazone and epirubicin,and zebrafish embryos were photographed 48 hours later.Results1.The docking results showed that PG-4C could bind to the active site of PPARγ target proteins.First,PG-4C forms a hydrogen bond with Phe282 of PPARγ.Secondly,almost all the loop structures weakly interact with the amino acids of PPARγ protein through van der Waals forces or weak bonds such as alkyl and π-alkyl groups.2.In vitro experiments showed that PG-4C could change the morphology of tumor cells,inhibit the proliferation of tumor cells,induce the apoptosis of gallbladder cancer cells,and affect the invasion and migration of tumor cells by disrupting the assembly and depolymerization of actin.3.PG-4C significantly inhibited the growth of gallbladder cancer in zebrafish and breast cancer in mice,and prolonged the survival period of the experimental animals.This indicated that PG-4C had a good drug activity.4.Drug toxicity test showed that PG-4C had lower toxicity and side effects than the positive control pioglitazone.5.Western Blot assay was used to verify the targeting activity of PG-4C to PPARy.By examining the Bcl-2 and Cleaved Caspase-3 signaling pathways,we found that PG-4C,when applied to tumor cells,resulted in increased PPARy content and altered expression of proteins important for apoptosis.Conclusions and innovationsThe aim of this study is to investigate the anti-gallbladder cancer and anti-breast cancer activity of a piperazine containing methyl glycyrrhetinic acid derivative PG-4C using PPARy protein as a drug target.The results showed that PG-4C had a significant effect on the morphology of gallbladder cancer cells and inhibited the proliferation of GBC-SD cells by arresting the cells in G2/M phase.In addition,PG-4C was able to induce apoptosis in GBC-SD cells.PG-4C was found to share and activate Caspases-3 by altering the ratio of cytoplasmic ROS to Ca2+and mitochondrial membrane potential,and activate the apoptotic signaling pathway.Western blot showed that the expression of Bcl-2 in GBC-SD cells was decreased after PG-4C treatment compared with control cells,and Caspase-3 was cleaved and activated.Meanwhile,PG-4C reduced the migration ability of GBC-SD cells by disrupting the microtubule network.In vivo experiments,PG-4C inhibited the growth of zebrafish GBC-SD xenograft tumor by 66%at the concentration of 10μM,and significantly inhibited the growth of breast cancer in mice at 20mg/kg.Demonstrated significant anticancer activity without damage to the experimental animals themselves.The above results indicate that PG-4C has the potential as an anticancer agent for gallbladder and breast cancer.This novel derivative provides a basis for the design of potent PPARy agonists and has great potential to improve disease management and treatment of cancer in the future.