Effect And Mechanism Of Ad-apoptin On Glycolysis And Metastasis Of Lung Adenocarcinoma Cells

Background: Lung cancer is one of the most common malignant tumors,in which non-small cell lung cancer(NSCLC)accounts for about 85%,lung adenocarcinoma is a non-small cell lung cancer,accounting for about 40% of primary lung tumors.The death of most patients with non-small cell lung cancer is related to metastasis,and epithelial mesenchymal transformation is an important cause of malignant tumor metastasis.Glycolysis plays an important role in the progression of lung adenocarcinoma.Enhanced glycolysis is an important factor to promote cell growth and metastasis.AMPK is a cellular energy receptor that is phosphorylated during energy stress.Low AMPK significantly enhanced the glycolysis of cancer cells,induced epithelial mesenchymal transition(EMT)markers and enhanced their invasion and migration.Mammalian target of rapamycin(m TOR)plays a key role not only in cell growth and proliferation,but also in cell proliferation and metabolism.AMPK phosphorylation inhibits m TOR activity,thereby inhibiting downstream targets P70S6K1 and 4ebp1,which is mainly involved in cell growth by regulating translation and protein synthesis.Apoptin is the product of VP3 gene of chicken anemia virus(CAV).It is a 13.6 k Da small protein,which can selectively kill a variety of human tumors or transformed cells,and has no toxic effect on normal cells.In previous studies,we constructed a viral vector expressing human Adenovirus type 5 apoptin and named it Ad-apoptin.Compared with traditional transfection reagents,this delivery system has been proved to effectively express apoptin protein in cells.However,the mechanism of Ad-apoptin inhibiting lung cancer is not clear.Objective: To investigate the effects of Ad-apoptin on proliferation,apoptosis,aerobic glycolysis,migration and invasion of lung adenocarcinoma cells,reveal the regulatory relationship between Ad-apoptin and AMPK,and further clarify the mechanism of Ad-apoptin inhibiting aerobic glycolysis and malignant progression of lung adenocarcinoma.Methods:(1)In vitro experiment: 1)The effect of Ad-apoptin on the proliferation of lung adenocarcinoma cells was determined by CCK-8 and crystal violet staining;2)JC-1staining,Annexin V-mcherry staining,annexin V-FITC/PI staining and Western blot were used to detect the effect of Ad-apoptin on the apoptosis of lung adenocarcinoma cells;3)The effects of Ad-apoptin on the migration and invasion of lung cancer cells were detected by wound experiment and Transwell chamber experiment;4)The effect of Ad-apoptin on EMT related marker protein of lung adenocarcinoma cells was detected by Western blot and cellular immunofluorescence staining;5)The expression levels of aerobic glycolysis related kinases in normal lung and lung cancer tissues were searched by GEPIA database and Kaplan-Meier database;6)The effects of Ad-apoptin on glucose uptake and lactate production in lung adenocarcinoma were detected by kit;7)The effects of Ad-apoptin on glycolytic key kinase and glucose transporter GLUT-1 in lung adenocarcinoma cells were detected by Western blot and cellular immunofluorescence staining;8)The interaction between Ad-apoptin and AMPK protein was detected by Co-IP experiment;9)When AMPK was knocked down,the effects of Ad-apoptin on glycolysis,migration and invasion of lung adenocarcinoma cells were detected.(2)In vivo experiments: 1)A stable AMPK gene knockdown cell line was constructed by lentivirus transfection;2)The subcutaneous tumor bearing model of nude mice was established to detect the effect of Ad-apoptin on tumor volume and survival rate of nude mice;3)The effect of Ad-apoptin on nude mice was detected by H&E staining;4)The effects of Ad-apoptin on the expression of Ki-67,TUNEL,glycolysis,migration and invasion related marker proteins in different groups of tumor tissues were detected by immunohistochemistry.Results:(1)In vitro experiment: 1)Ad-apoptin could inhibit the proliferation of lung adenocarcinoma cells in a dose-dependent and time-dependent manner by CCK-8 and crystal violet staining;2)The results of JC-1 staining,Annexin V-mcherry staining and annexin V-FITC/PI staining showed that Ad-apoptin induced apoptosis in non-small cell lung cancer.Western blot showed that Ad-apoptin induced the up regulation of apoptotic proteins cleaved PARP and cleaved caspase-3;3)Western blot showed that Ad-apoptin could induce the down-regulation of Vimentin,Fibronectin,N-cadherin,Snail and MMP9 proteins and up regulate the expression of E-cadherin protein in lung adenocarcinoma cells.The results of cellular immunofluorescence were consistent with them;4)Through the search of GEPIA database and Kaplan-Meier database,it was found that the key glycolytic kinases PKM2,LDHA,HK2 and GLUT-1 were highly expressed in lung cancer,which was negatively correlated with the survival rate;5)Ad-apoptin inhibited glucose uptake and lactate production in lung adenocarcinoma cells.Western blot showed that Ad-apoptin inhibited the expression of glycolytic key proteins(HK1,HK2,PKM1,PKM2 and LDHA)and glucose transporter GLUT-1;6)Ad-apoptin decreased the expression of p-m TOR,p-p70S6 K and p-4E-BP1 protein and up regulated the expression of p-AMPk protein in lung adenocarcinoma cells;Co-IP results showed that exogenous apoptin was precipitated by AMPK,which was the target gene of apoptin;7)Knockdown of AMPK can weaken the ability of Ad-apoptin to inhibit glycolysis,migration and invasion of non-small cell lung cancer cells.(2)In vivo experiments: 1)a Ad-apoptin treatment significantly reduced the tumor volume and prolonged the survival time of mice;2)H&E results showed that in Ad-apoptin group,karyolysis and pyknosis were observed,and vacuoles in cytoplasm increased;Immunohistochemical results showed that compared with Control group and Ad-mock group,the positive rate of Ki-67 in Ad-apoptin group decreased,while the positive rate of TUNEL increased;The expressions of p-4E-BP1,N-cadherin,GLUT-1 and LDHA decreased in Ad-apoptin group;On the contrary,the expression of p-AMPK and E-cadherin increased,which was consistent with the results in vitro;3)The results of Western blot and q RT-PCR showed that the stable transformation of A549 cell line sh AMPK was successfully constructed.In the subcutaneous transplantation models of nude mice in different groups,there were significant differences in tumor volume and survival rate between sh AMPK + Ad-apoptin group and Ad-apoptin group.Compared with Ad-apoptin group,the positive rate of Ki-67 and the expression of p-AMPK in sh AMPK +Ad-apoptin group were higher,while the positive rate of TUNEL and the expression of p-m TOR were lower,which was consistent with the results in vitro.Conclusion: Ad-apoptin can activate AMPK/m TOR signaling pathway by targeting AMPK,and then inhibit the proliferation,migration,invasion and glycolysis of lung adenocarcinoma cells.In addition,we found that in the subcutaneous transplantation model of nude mice,intraperitoneal injection of Ad-apoptin can significantly reduce the tumor volume and prolong the survival time of nude mice.