title: Combination treatment with erlotinib and ampelopsin overcomes erlotinib resistance in NSCLC cells via the Nox2-ROS-Bim pathway.
abstract: Erlotinib, a tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), has been shown to have a dramatic effect in non-small cell lung cancer (NSCLC) patients with EGFR mutation. However, the presence of primary resistance or acquired resistance to EGFR-TKI is the most common reason for switching to other anti-cancer agents. Even though there are newer agents that have activity in the presence of the T790M mutation, identification of potential agents that could overcome resistance to EGFR-TKI is still needed for the treatment of NSCLC patients.
  
  
  
  In this study, we used erlotinib-resistant NSCLC cell lines to investigate the effects of combination treatment with erlotinib and ampelopsin. After treatment with either single or combination, cell viability and cell death were determined with WST-1 assay, trypan blue exclusion method, colony forming assay, annexin-V staining assay and western blot assay. The content of ROS was evaluated by FACS analysis using H2DCF-staining method. To determine the effect of Nox2 and Bim on the combined treatment with erlotinib and ampelopsin-induced cell death, we transfected with Nox2 or Bim specific siRNA and performed with western blot assay for evaluation of its expression.
  
  
  
  Combined treatment with erlotinib and ampelopsin at non-cytotoxic concentrations significantly induced caspase-dependent cell death in erlotinib-resistant NSCLC cells. Furthermore, cell death resulted in the accumulation of reactive oxygen species (ROS) through upregulation of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) expression, a direct source of ROS. The expression level of Bim increased with combination treatment, but not with either treatment alone.
  
  
  
  Here in this study, we demonstrate that the combination of erlotinib and ampelopsin induces cell death via the Nox2-ROS-Bim pathway, and ampelopsin could be used as a novel anti-cancer agent combined with EGFR-TKI to overcome resistance to erlotinib in EGFR-mutant NSCLC.
authors.affiliation: Department of Anatomy, Inje University College of Medicine, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea.
authors.forename: Seung-Woo
authors.initials: SW
authors.lastname: Hong
authors.affiliation: Department of Anatomy, Inje University College of Medicine, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea.
authors.forename: Nam-Sook
authors.initials: NS
authors.lastname: Park
authors.affiliation: Department of Anatomy, Inje University College of Medicine, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea.
authors.forename: Min Hye
authors.initials: MH
authors.lastname: Noh
authors.affiliation: Department of Anatomy, Inje University College of Medicine, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea.
authors.forename: Ju A
authors.initials: JA
authors.lastname: Shim
authors.affiliation: Department of Anatomy, Inje University College of Medicine, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea.
authors.forename: Byul-Nim
authors.initials: BN
authors.lastname: Ahn
authors.affiliation: Department of Anatomy, Inje University College of Medicine, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea.
authors.forename: Yeong Seok
authors.initials: YS
authors.lastname: Kim
authors.affiliation: Department of Anatomy, Inje University College of Medicine, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea.
authors.forename: Daejin
authors.initials: D
authors.lastname: Kim
authors.affiliation: Devision of Pulmonology, Department of internal Medicine, Inje University Pusan Paik Hospital, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea. Electronic address: goodoc@inje.ac.kr.
authors.forename: Hyun-Kyung
authors.initials: HK
authors.lastname: Lee
authors.affiliation: Department of Anatomy, Inje University College of Medicine, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea. Electronic address: dyhur@inje.ac.kr.
authors.forename: Dae Young
authors.initials: DY
authors.lastname: Hur
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor / Q000187:drug effects; D002986:Clinical Trials as Topic; D003131:Combined Modality Therapy / Q000379:methods*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics; D000069347:Erlotinib Hydrochloride / Q000378:metabolism / Q000494:pharmacology*; D005419:Flavonoids / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D000074662:NADPH Oxidase 2 / Q000235:genetics*; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D005419:Flavonoids; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C106407:ampelopsin; D000069347:Erlotinib Hydrochloride; D000074662:NADPH Oxidase 2; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Ampelopsin; Erlotinib; NSCLC; Nox2; ROS