Recombinant Newcastle Disease Virus Expressing IL-29 (rL-29)-induced Apoptosis Of A549 Cells Is Connected To Endoplasmic Reticulum Stress Pathways

Objective:IL-29 is a cytokine consisting of anti-tumor and anti-proliferative actions.Recent studies have showed that recombinant Newcastle disease virus(NDV)expressing IL-29(named as r L-29)plays a role in apoptosis of gastric cancer cells.It was also reported that endoplasmic reticulum stress(ERS)induced autophagy and apoptosis in tumor cells.In this study,we aimed to explore the relationship and underlying mechanism between endoplasmic reticulum stress and r L-29-induced apoptosis of lung adenocarcinoma cells A549.Methods:1.Infection efficiency of virus Western Blot was used to identify the level of IL-28 R in A549,SK-MES-1 and Lewis cell lines;ELISA kit,PCR,immunofluorescence were used to examine the level of IL-29 after infected with virus.2.Effect of r L-29 on A549 cell proliferation and migration A549 cells were infected with various MOI of r L-29,NDV for 24 h,MTT was performed to assess cell inhibition rate;The inhibitory properties of r L-29 on A549 cells were also determined by a clonogenic assay;Transwell and scratch migration assays were used to explore the capability of migration.3.ERS response and apoptosis in A549 cells induced by r L-29 We measured the levels of endoplasmic reticulum stress associated proteins,such as GRP78,CHOP,p-e IF2? in A549 cells that were infected with r L-29 by Western blot;Immunofluorescence imaging was used to measure the expression of GRP78 and CHOP;Western Blot was performed to measure the levels of apoptosis associated proteins,such as cleaved caspase3,cleaved caspase8,cleaved caspase9,BCL-2/bax.4.Effect of blockage of ER stress on r L-29 triggered autophagy and apoptosis in A549 cells First,A549 cells were administrated with r L-29,NDV and PBS in the presence or absence of 4-phenylbutyrate(4-PBA)or si RNA CHOP or si RNA PERK.Then,Western Blot was performed to measure the expression levels of Endoplasmicreticulum stress,apoptosis and autophagy related proteins.5.Effect of JNK inhibitor on apoptosis and autophagy A549 cells were administrated with r L-29,NDV and PBS for 24 h at an MOI of10 in the absence or presence of the JNK inhibitor SP600125.Western Blot was used to measure the levels of p-JNK,LC3 II,cleaved caspase3.6.Effect of BCL-2 knockdown on autophagy and apoptosis FAM-si RNA of BCL-2 was utilized to determine transfection efficiency of si BCL-2.Another,colony formation numbers were measured by clonal clusters stained with crystal violet.Furthermore,Western Blot was performed to identify several proteins involved in autophagy(LC3II,beclin1)and apoptosis(cleaved caspase3).7.Inhibitory effects of r L-29 on subcutaneous growth of A549 cells in vivo and partial mechanisms First,tumour-bearing mouse model was constructed.Then r L-29,NDV and PBS were injected in the tumor and the tumour volume was measured regularly.Next,Western Blot was used to investigate ERs associated proteins(GRP78,CHOP,p-e IF2?),autophagy associated proteins(beclin1,LC3II),apoptosis associated proteins(cleaved caspase3).Results:1.In this study,A549 cell lines displayed higher levels of IL-28 R expression than the SK-MES-1 and Lewis lines.ELISA kit,PCR,immunofluorescence showed that r L-29 was successfully infected in the r L-29 group.2.Cell viability was effectively inhibited by r L-29 and NDV at an MOI of 10.Compared with NDV and PBS,r L-29 greatly reduced colony formation by clonogenic survival assay.Transwell assay and scratch experiment revealed that A549 cells infected with r L-29 migrated slower than the NDV and PBS groups.3.Compared with the NDV and PBS groups,Western Blot was used to show that the levels of ERS related proteins(GRP78,CHOP,p-e IF2?)and apoptosis related proteins(cleaved caspase3,cleaved caspase8 and cleaved caspase9)upregulated in A549 cells that were treated with r L-29,while apoptosis related proteins BCL-2/Bax decreased.Immunofluorescence imaging measured the expression of ERS markers such as GRP78 and CHOP significantly elevated in the r L-29group.4.Western Blot was performed to detect that the expression of Endoplasmic reticulum stress,apoptosis and autophagy related proteins decreased dramatically with administration of 4-phenylbutyrate(4-PBA)or si RNA CHOP or si RNA PERK.5.Western Blot was used to found that the expression of p-JNK,cleaved caspase3 and LC3 II significantly attenuated in the presence of the JNK inhibitor.6.FAM-si RNA of BCL-2 was utilized and found that si BCL-2 was successfully transfected;Another,we observed that si BCL-2 reduced the colony formation numbers of A549.Furthermore,Western Blot was used to found that a significant increase in the expression of LC3 II,beclin1 and cleaved caspase3 after treatment of si RNA-mediated BCL-2.Conclusion:r L-29 inhibited A549 cell proliferation and migration and induced ERS,autophagy and apoptosis in A549 cells and tissues after treated with r L-29.Further,r L-29 induced autophagy and apoptosis of A549 cells via PERK-e IF2? pathway of endoplasmic reticulum stress response and IRE1/JNK/BCL-2 pathways.