Study designs to investigate Nox1 acceleration of neoplastic progression in immortalized human epithelial cells by selection of differentiation resistant cells.
To investigate the role of NADPH oxidase homolog Nox1 at an early step of cell transformation, we utilized human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus (HPV) type 16 (GM16) to generate progenitor cell lines either by chronic ethanol exposure or overexpression with Nox1. Among several cobblestone epithelial cell lines obtained, two distinctive spindle cell lines - FIB and NuB1 cells were more progressively transformed exhibiting tubulogenesis and anchorage-independent growth associated with increased invasiveness. These spindle cells acquired molecular markers of epithelial mesenchymal transition (EMT) including mesenchymal vimentin and simple cytokeratins (CK) 8 and 18 as well as myogenic alpha-smooth muscle actin and caldesmon. By overexpression and knockdown experiments, we showed that Nox1 on a post-translational level regulated the stability of CK18 in an ROS-, phosphorylation- and PKCepilon-dependent manner. PKCepilon may thus be used as a therapeutic target for EMT inhibition. Taken together, Nox1 accelerates neoplastic progression by regulating structural intermediate filaments leading to EMT of immortalized human gingival epithelial cells.
School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand.
Apsorn
A
Sattayakhom
School of Allied Health Sciences and Public Health, Walailak University, Nakhon Si Thammarat, Thailand.
Warangkana
W
Chunglok
Faculty of Medical Technology, Mahidol University, Bangkok, Thailand.
Wanida
W
Ittarat
Department of Internal Medicine IV, University Heidelberg Hospital, Heidelberg, Germany.
Walee
W
Chamulitrat
D002454:Cell Differentiation / Q000187:drug effects; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000473:pathology*; D018450:Disease Progression; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology* / Q000821:virology; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D000431:Ethanol / Q000494:pharmacology; D005881:Gingiva; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007382:Intermediate Filaments / Q000502:physiology; D053538:Keratin-18 / Q000378:metabolism; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000502:physiology*; D009361:Neoplasm Invasiveness; D009363:Neoplasm Proteins / Q000502:physiology*; D009856:Oncogene Proteins, Viral / Q000502:physiology; D050725:Papillomavirus E7 Proteins / Q000502:physiology; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000502:physiology; D011499:Protein Processing, Post-Translational; D017382:Reactive Oxygen Species; D012097:Repressor Proteins / Q000502:physiology; D012641:Selection, Genetic
C068651:E6 protein, Human papillomavirus type 16; D007381:Intermediate Filament Proteins; D053538:Keratin-18; D009363:Neoplasm Proteins; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D000431:Ethanol; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; D051744:Protein Kinase C-epsilon
AIG, anchorage-independent growth; CK, cytokeratin; Cobblestone cells; Cytokeratins; EGF, epidermal growth factor; EMT; EMT, epithelial mesenchymal transition; GM, gingival mucosal; HPV, human papillomavirus; IAP, inhibitor of apoptosis protein; Immortalized gingival keratinocytes; Intermediate filaments; Invasion; MEF2, myocyte enhancing factor 2; MMP, matrix metalloproteinases; Nox, NAD(P)H oxidase; PMA, 12-O- tetradecanoylphorbol-13-acetate; ROS, reactive oxygen species; Spindle cells; iNOS, inducible nitric oxide synthase