title: Chronic exposure of lung alveolar epithelial type II cells to tobacco-specific carcinogen NNK results in malignant transformation: a new in vitro lung carcinogenesis model.
abstract: Lung cancer is the leading cause of cancer-related mortality in both men and women throughout the world. This disease is strongly associated with tobacco smoking. The aim of this manuscript was to establish an in vitro model that mimics the chronic exposures of alveolar epithelial type II cells to the tobacco-specific nitrosamine carcinogen, NNK. Immortalized non-neoplastic alveolar epithelial cells type II, (E10 cells), from BALB/c mice were exposed to low concentration of NNK (100 pM) during 5, 10, 15, and 20 cycles of 48 h. NNK-transformed cells showed an increase of proliferation rate and motility. Moreover, these cells underwent epithelial-to-mesenchymal transition (EMT). Increased migratory capacity and EMT were correlated to the time of exposure to NNK. NNK-transformed cells were tested for their growth and metastatic capacity in vivo. Subcutaneous injection of cells exposed to NNK for 20 cycles (E10-NNK20 clone) into BALB/c mice led to the formation of subcutaneous tumors that arose after 40 ± 17 d in all animals, which died 95 ± 18 d after cell inoculation, with lymph nodes and lung metastasis. The morphological characteristics of tumors were compatible with metastatic undifferentiated carcinoma. Cells exposed to NNK for 5-10 cycles did not display metastatic capacity, while those exposed for 15 cycles displayed low capacity. Our results show that prolonged exposures to NNK led the cells to increasingly acquire malignant properties. The cellular model presented in this study is suitable for studying the molecular events involved in the different stages of malignant transformation.
authors.affiliation: Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, São Paulo, SP, Brazil.
authors.forename: Gregory
authors.initials: G
authors.lastname: Mennecier
authors.forename: Luciana N
authors.initials: LN
authors.lastname: Torres
authors.forename: Bruno
authors.initials: B
authors.lastname: Cogliati
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Sanches
authors.forename: Claudia M
authors.initials: CM
authors.lastname: Mori
authors.forename: Andréia O
authors.initials: AO
authors.lastname: Latorre
authors.forename: Lucas M
authors.initials: LM
authors.lastname: Chaible
authors.forename: Ivone I
authors.initials: II
authors.lastname: Mackowiak
authors.forename: Marcia K
authors.initials: MK
authors.lastname: Nagamine
authors.forename: Tereza C
authors.initials: TC
authors.lastname: Da Silva
authors.forename: Heidge
authors.initials: H
authors.lastname: Fukumasu
authors.forename: Maria Lucia Zaidan
authors.initials: ML
authors.lastname: Dagli
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002273:Carcinogens / Q000633:toxicity*; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009362:Neoplasm Metastasis; D009602:Nitrosamines / Q000633:toxicity*; D011650:Pulmonary Alveoli / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D014945:Wound Healing; D014026:Nicotiana
chemical_list: D002273:Carcinogens; D009602:Nitrosamines; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone