Glycyrrhetinic acid-triggered cytoprotective autophagy via inositolrequiring enzyme 1α-c-Jun N-terminal kinase cascade in non-small cell lung cancer cells

OBJECTIVE Glycyrrhetinic acid(GA),one of the main bioactive constituents of the famous Chinese medicinal herb Glycyrrhizauralensis Fisch,presents potent cytotoxicity to various cancer cells both in vitro and in vivo.Herein,we′d like to determine whether GA triggers autophagy in non-small cell lung cancer cells and the mechanisms involved.METHODS Cell proliferation was determined by MTT assay and colony formation.AnnexinⅤ/PI staining,Hoechst33342 staining,and Western blotting were used to detect GA-induced apoptosis.GA-induced autophagy was measured by expression of the lipid modification of light chain-3(LC3)and transfected with GFP-LC3 or GFP-RFP-LC3 plasmid.Pharmacological regulators,siRNA,and plasmid transfection were used to study the mechanisms of GA-triggered autophagy.RESULTS GA inhibited cell proliferation and induced apoptosis in a concentrationdependent manner in non-small cell lung cancer A549 cells.GA induced autophagyas evidenced by up-regulation of LC3-Ⅱexpression when combined treatment with chloroquine and induction of the red punta after GFP-RFP-LC3 plasmid transfection.Knockdown of autophagy related proteins(ATG)7,ATG 5,or beclin 1by siRNA,the expression of LC3-Ⅱand GFP-LC3 punt atriggered by GA were decreased.Furthermore,the c-Jun N-terminal kinase(JNK)pathways were activated after treatment with GA,and pretreatment with JNK inhibitor SP600125 or silence of JNK pathway by siRNA of JNK or c-jun obviously reduced GA-induced LC3-Ⅱexpression and GFP-LC3 punta formation.GA also stimulate dendoplasmic reticulum stress response by triggering inositol-requiring enzyme 1α(IRE1α)pathway,and knockdown of IRE1αinhibited the activation of JNK pathway and autophagy induced by GA.In addition,GA-induced cell proliferative inhibition and apoptosis were both enhanced when silence of autophagy as well as JNK pathway.CONCLUSION Our study demonstrated,for the first time,that GA induced a cytoprotective autophagy in non-small cell lung cancer cells by activating the IRE1α-JNK pathway,which might decreased the anti-cancer effects of GA.