The Regulatory Function Of Peroxiredoxin Prx Ⅴ On Apoptosis Of Liver Cancer Cells Induced By Ethyl-β-carboline-3-carboxylate

Primary liver cancer is one of the most common cancers in the world.At present,Chinese herbal extracts are widely used in the treatment of various diseases due to their strong efficacy and easy extraction.Ethyl-β-carboline-3-carboxylate(β-CCE)is a β-carboline compound of picrasma quassioides extract,which has various drug activities such as anti-inflammatory,antibacterial,treatment of neurological diseases and anticancer.However,the research on its anticancer pharmacological activity is not perfect.Peroxidase V(Peroxiredoxin V,Prx Ⅴ)is a member of the Prxs family,widely distributed in various types of cells,especially in mitochondria and cytoplasm with strong antioxidant capacity,by decomposing excessive intracellular substances.Reactive oxygen species(Reactive Oxigen Species,ROS)and reactive nitrogen species(Reactive Ntirogen Oxide Species,RNS),maintain intracellular redox homeostasis.In this study,aiming at the characteristics that Prx Ⅴ can effectively regulate the level of intracellular ROS,by constructing a Hep G2 cell line overexpressing Prx Ⅴ,to explore the regulation and effect of Prx Ⅴ on the apoptosis of hepatocellular carcinoma cells in the process of β-CCE-induced apoptosis of Hep G2 cells This mechanism provides an effective target for β-CCE clinical liver cancer treatment,and provides a new theoretical basis for revealing the regulatory role of Prx Ⅴ in liver cancer chemotherapy.To evaluate the toxic effect of β-CCE on hepatoma cells and the effect on the expression of Prx Ⅴ protein.The sensitivity of three types of hepatoma cells to β-CCE was detected by MTT method,the effect of β-CCE on the survival rate of Hep G2 cells at concentration/time intervals was detected,and the cytotoxicity of β-CCE on two normal hepatocytes was detected at the same time;Violet staining was used to detect the effect of β-CCE on the colony formation ability of Hep G2 cells.The effect of β-CCE on the migration ability of Hep G2 cells was detected by cell scratch assay.The expression levels of reactive oxygen species in Hep G2 cells treated withβ-CCE and in mitochondria were detected by fluorescence microphotography,and mitochondrial damage and cell viability after drug treatment were detected.Western blotting was used to detect the changes of β-CCE on the expression of apoptosis proteins,Prxs protein family and MAPK signaling pathway in Hep G2 cells.In order to use Prx Ⅴ overexpression and the Mock group Hep G2 cell line to elucidate the regulatory role and mechanism of Prx Ⅴ in the process of β-CCE-induced hepatocellular carcinoma cell apoptosis,bioinformatics software was used to analyze the expression of Prx Ⅴ in hepatocellular carcinoma and normal tissues.The difference of expression and the effect on the survival rate of liver cancer patients suggested the relationship between Prx Ⅴ and the occurrence,development and prognosis of clinical liver cancer.Then,Prx Ⅴ overexpression and Mock(control)cell lines were constructed by lentiviral vector,and Prx Ⅴ overexpression Hep G2 cells constructed by lentiviral vector and the Hep G2 cell line in Mock group were successfully constructed by Western boltting detection.The effect of β-CCE on the cell viability of Mock &Prx Ⅴ overexpressing Hep G2 cells was detected and proved by MTT method,and the intracellular and mitochondrial reactive oxygen species levels in Mock&Prx Ⅴ overexpressing Hep G2 cells treated with β-CCE were compared by fluorescence microscopy.The differences were compared,and the mitochondrial damage and cell activity changes were compared.Western blotting was used to detect the expression levels of apoptosis-related proteins and the changes of MAPK signaling pathway in Mock & Prx Ⅴ overexpressed Hep G2 cells by β-CCE.The results showed that β-CCE could effectively reduce the survival rate of Hep G2 cells and inhibit the ability of cell colony formation and migration.After screening the drug concentration,80 μg/m L and 100 μg/m L were selected for follow-up experiments.It was found that at this concentration,β-CCE could significantly increase the accumulation of reactive oxygen species in cells and mitochondria,and at the same time induce a decrease in mitochondrial membrane potential.In addition,by investigating the expression levels of apoptosis-related proteins,it was found that the mitochondrial-dependent apoptosis protein cleavage-Caspase 9 and the pro-apoptotic proteins Bad,Bax and cleavage-Caspase 3 were significantly increased,and the anti-apoptotic proteins Bcl2 and Bcl-x L were significantly increased.The expression level was significantly decreased,which proved that β-CCE induced apoptosis of Hep G2 cells by increasing the level of intracellular reactive oxygen species and damaging cell mitochondria.The analysis of the signal transduction pathway showed that β-CCE could effectively affect the MAPK signal pathway,reduce the phosphorylation level of ERK protein,and increase the phosphorylation level of JNK and P38 protein.When the reactive oxygen species scavenger N-acetyl-L-cysteine(NAC)pretreated cells,it could effectively reduce the increase of intracellular and mitochondrial reactive oxygen species in Hep G2 caused byβ-CCE,and at the same time caused a significant increase in the mitochondrial membrane potential of cells decreased,decreased the level of apoptosis,and down-regulated the activation of MAPK signaling pathway.It was found that the expression of Prx Ⅴ protein in Hep G2 cells decreased with drug treatment,but NAC pretreatment could inhibit the down-regulation of Prx Ⅴ protein expression induced by β-CCE.By using β-CCE to treat Mock & Prx Ⅴ overexpressing Hep G2 cells,it was found that the survival rate of Hep G2 cells in the Prx Ⅴ overexpression group was higher than that in the Mock group,and the intracellular and mitochondrial reactive oxygen species levels in the Prx Ⅴ group were lower than those in the Mock group.The mitochondrial membrane potential of group was higher than that of Mock group.In addition,overexpression of Prx Ⅴ effectively reduced β-CCE-induced apoptosis,and at the same time effectively down-regulated the activation of MAPK signaling pathway.After treating the two groups of cells with NAC,the levels of intracellular and mitochondrial reactive oxygen species and mitochondrial damage in the two groups were significantly decreased,the apoptosis was significantly reduced,and the activation of the MAPK signaling pathway was significantly reversed.These results show that overexpression of Prx Ⅴ can effectively inhibit the apoptosis of Hep G2 hepatoma cells induced by β-CCE,and scavenging of reactive oxygen species is a key factor for Prx Ⅴ to exert these effects.In conclusion,Prx Ⅴ plays an important regulatory role in the apoptosis of Hep G2 hepatoma cells induced by β-CCE.Its mechanism of action is mainly to effectively remove intracellular reactive oxygen species,reduce mitochondrial damage,down-regulate ROS-dependent MAPK signaling pathway and apoptosis-related proteins,and finally reduce the mitochondria-dependent apoptosis of Hep G2 liver cancer cells mediated by β-CCE.This study clarifies the mechanism of Prx Ⅴ in β-CCE-induced apoptosis of liver cancer cells,and provides a feasible basis for gene therapy of liver cancer and a new theoretical basis for the development of chemotherapy drugs for liver cancer.