title: Inhibition of cytoplasmic GSK-3β increases cisplatin resistance through activation of Wnt/β-catenin signaling in A549/DDP cells.
abstract: Cisplatin-based chemotherapy is recommended as the first-line therapy for advanced non-small cell lung cancer (NSCLC). However, acquired cisplatin resistance is ubiquitous in patients with NSCLC, but the molecular mechanism of such resistance remains ambiguous. The present study sought to examine the role of the Wnt/β-catenin signaling pathway in cisplatin resistance by assessing the phosphorylation and subcellular distribution of GSK-3β in a human lung adenocarcinoma cell line, A549, and its cisplatin-resistant subline, A549/DDP. Total GSK-3β, phosphorylated GSK-3β(ser9) and phosphorylated GSK-3β(tyr216) in cytoplasmic and nuclear fractions of A549/DDP and A549 cells were examined by western blot analysis. The regulation of cisplatin resistance, apoptosis, β-catenin and survivin protein expression by inhibition of cytoplasmic GSK-3β were determined by MTT assay, flow cytometry analysis, immunofluorescence technique and western blot analysis. In the present study, cytoplasmic levels of p-GSK-3β(ser9) were significantly increased in A549/DDP cells as compared with A549 cells (P<0.01), and these levels were further increased by cisplatin treatment in A549/DDP cells (P<0.01). In contrast, cytoplasmic levels of p-GSK-3β(ser9) were reduced in A549 cells after treatment with cisplatin (P<0.01). However, cytoplasmic levels of p-GSK-3β(tyr216) were significantly decreased in A549/DDP cells as compared with A549 cells (P<0.01), and these levels were further decreased by cisplatin treatment in A549/DDP cells (P<0.01). Conversely, cytoplasmic levels of p-GSK-3β(tyr216) were raised in A549 cells after treatment with cisplatin (P<0.01). Analysis of downstream effectors of the Wnt/β-catenin signaling pathway revealed upregulation of β-catenin and survivin expression in A549/DDP cells treated with cisplatin as compared to untreated cells. In A549 cells, cisplatin treatment decreased the expression of β-catenin and survivin. Furthermore, phosphorylation of GSK-3β at serine 9 by LiCl and transient interference of GSK-3β by siRNA increased β-catenin and survivin protein expression in A549/DDP cells. Low exogenous and endogenous cytoplasmic GSK-3β expression enhanced the IC50 and inhibited apoptosis. In conclusion, activation of the Wnt/β-catenin signaling pathway and upregulated survivin expression due to cytoplasmic GSK-3β inhibition might lead to cisplatin resistance in NSCLC.
authors.affiliation: Department of Pathology, The First Affiliated Hospital and College of Basic Medical Sciences of China Medical University, Shenyang 110001, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.forename: Zan
authors.initials: Z
authors.lastname: Liu
authors.forename: Xiuwei
authors.initials: X
authors.lastname: Zhang
authors.forename: Jiani
authors.initials: J
authors.lastname: He
authors.forename: Yongqi
authors.initials: Y
authors.lastname: Pan
authors.forename: Fengxia
authors.initials: F
authors.lastname: Hao
authors.forename: Lingling
authors.initials: L
authors.lastname: Xie
authors.forename: Qingchang
authors.initials: Q
authors.lastname: Li
authors.forename: Xueshan
authors.initials: X
authors.lastname: Qiu
authors.forename: Enhua
authors.initials: E
authors.lastname: Wang
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002945:Cisplatin / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D020128:Inhibitory Concentration 50; D034741:RNA, Small Interfering / Q000378:metabolism; D000077022:Survivin; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism
chemical_list: C107489:BIRC5 protein, human; C495270:CTNNB1 protein, human; D051034:Inhibitor of Apoptosis Proteins; D034741:RNA, Small Interfering; D000077022:Survivin; D051176:beta Catenin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D002945:Cisplatin