Regulatory Effect And Mechanism Of Prx Ⅴ On Oxidative Stress Response In Apoptosis Of Gastric Cancer Cells Induced By Antitumor Drugs

As one of the four major cancers causing human death,Gastric cancer is a disease that seriously threatens human health and life.Because the early symptoms are not obvious,most patients are in the late stage after diagnosis,which brings great difficulties to clinical treatment.Cell therapy and supportive care.However,there are still many problems in the clinical treatment of gastric cancer due to the high risk and recurrence of surgical Treatment,such as irreversible damage to normal tissues caused by radiotherapy,toxic side effects of chemical drugs,high cost and immature technology of cell therapy.In recent years,changing the local microenvironment of cancer has become a hot research topic.The physiological activity of tumor microenvironment is very different from that of human normal internal environment.The core part of tumor tissue is often exposed to hypoxia or hypoxia for a long time due to a large number of cell proliferation,resulting in a sharp rise in the level of ROS in cancer cells.Intracellular ROS can regulate cell proliferation and metabolism,which are closely related to tumorigenesis and treatment.Therefore,effectively regulating the level of ROS in cancer cells and inducing apoptosis of cancer cells is a new breakthrough and research idea in the treatment of gastric cancer.Peroxiredoxin Ⅴ(Prx Ⅴ)can reduce hydrogen peroxide,alkyl hydroperoxide and peroxynitrite,clear intracellular ROS,maintain intracellular redox balance,reduce the oxidative damage caused by intracellular oxidative stress,and then activate related downstream signaling pathways to regulate apoptosis.As a member of the peroredoxin(Prxs)family,it is widely distributed in the cytoplasm,mitochondria and peroxisome,and abnormal expression in a variety of cancers.Therefore,in this study,we used emodin and doxorubicin hydrochloride,two drugs that can increase intracellular ROS level,to treat human gastric cancer AGS cell line which can induce apoptosis of AGS cells by increasing intracellular ROS level.At the same time,we used lentiviral vector to construct the silencing or overexpression Prx Ⅴ AGS cells to understand the regulatory role of PrXV in apoptosis of gastric cancer cells induced by high ROS level and its possible molecular mechanism.Part Ⅰ Peroxiredoxin Ⅴ inhibits emodin induced apoptosis of gastric cancer cells through ROS/BCL2 signaling pathwayObject:To investigate the regulatory role of Prx Ⅴ in emodin induced apoptosis of gastric cancer cell line AGS.Methods:ROS levels in AGS cells treated with emodin at different times were detected by flow cytometry with DCFH-DA staining.Annexin-V-FITC and PI double staining was used to detect AGS cell apoptosis treated with emodin for different time by fluorescence microscopy and flow cytometry.Western blot was used to detect the expression of Prxs family proteins in AGS cells treated with emodin for different time.After pretreatment of cells with ROS inhibitor NAC,the apoptosis was detected by flow cytometry,Western blot detection of Prxs family protein expression.Lentiviral vector transfection of AGS cell line,and stable genetic blank vector group(mock),Prx Ⅴ silencing group(shprx V)and Prx Ⅴ overexpression group(Prx Ⅴ his)cell lines were constructed.Western blot detection of protein expression level.Flow cytometry was used to detect the effects of emodin on three kinds of cell apoptosis and intracellular ROS under different time and concentration.Western blot detection of apoptosis expression related proteins in three kinds of cells treated with emodin at different time.Results:Emodin can significantly reduce the survival rate of AGS cells,and significantly increase the level of cellular reactive oxygen species,in a time-and dose-dependent manner.Western blot results showed that the expression of prxv in the process of apoptosis is significantly different.At the same time,overexpression of Prx Ⅴ can down-regulate the expression of pro-apoptotic proteins Bad and cleaved PARP,and up-regulate the expression level of anti-apoptotic protein BCL2 in AGS cells.After treatment with active oxygen scavengers,emodin can significantly reduce the level of active oxygen and apoptosis of AGS cells.Conclusion:1.Emodin can induce AGS cell apoptosis by increasing intracellular ROS level.2.With the increase of emodin treatment time,the expression level of Prx Ⅴ protein in AGS cells is significantly reduced.3.The expression of Prx Ⅴ can regulate the apoptosis of AGS cells induced by emodin.Part Ⅱ Silencing peroxiredoxin V gene enhances doxorubicin induced mitochondrion dependent apoptosis in gastric cancer cellsObjective:To explore the possible regulatory effect and mechanism of Prx Ⅴ on doxorubicin induced apoptosis of AGS gastric cancer cells.Methods:Lentiviral vectors were transfected into AGS cell lines to construct stable genetic blank vector group(mock)and Prx Ⅴ silence group(shprx V)cell lines.The MTT method was used to detect the survival rate of the two cells treated with different concentrations of doxorubicin(DOX).Annexin V-PE staining,flow cytometry and fluorescence microscopy were used to detect the apoptosis of the two kinds of cells after treatment with adriamycin at different times and concentrations.DHE staining,flow cytometry and fluorescence microscopy were used to detect the ROS production of two kinds of cells after treatment with different concentrations of DOX.Flow cytometry and fluorescence microscopy detect the production of mitochondrial ROS.JC-1 staining was used to detect the change of mitochondrial membrane potential,and Western blot was used to detect the expression of apoptosis-related proteins.Results:Prx Ⅴ gene silencing could significantly increase DOX induced apoptosis by increasing intracellular ROS accumulation.We also found that DOX induced changes in mitochondrial ROS level and membrane permeability in shprx V cells were significantly higher than those in mock cells,which were significantly reversed by NAC treatment.Prx Ⅴ gene silencing also significantly up-regulated the expression of cleaved caspase-9,3 and down-regulate the BCL2 related proteins.Conclusion:1.Silencing the expression of Prx Ⅴ in AGS cells can increase the drug sensitivity to DOX treatment.2.Silencing the expression level of Prx Ⅴ in cells can increase the accumulation of ROS in cells and mitochondria.3.Pretreatment of NAC can effectively alleviate Dox induced apoptosis.