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.

Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier

“…This is different from carcinoma-derived cell lines, which are beneficial for studying cancer development, e.g., a blastoma-derived cell line (Camerlingo et al, 2011) or an in vitro carcinogenesis model, where ATII cells are exposed to tobacco-specific carcinogens (Mennecier et al, 2014) but hardly represent normal epithelial cells. The widely used A549 cell line, derived from an adenocarcinoma, which is commonly employed as a model of the lung epithelium, has a rather ATIIlike phenotype, including lamellar bodies (Shapiro et al, 1978).…”

Long‐term exposure to extremely low‐dose of nicotine and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) induce non‐malignant breast epithelial cell transformation through activation of the a9‐nicotinic acetylcholine receptor‐mediated signaling pathway

“…During the tumor progression stage and at a stage before an acquisition of invasive ability, cancerous cells undergo adaptation and plasticity to enhance the cell mobility. A previous study demonstrated that lung epithelial cells chronically exposed to NNK at 100 picomolar concentration acquired migration ability in the wound healing assay. To test whether the HBL‐100 breast epithelial cells induced by exposure to the SHS doses were able to acquire migration ability, the transwell migration and wound healing assays were performed.…”

“…In the current study, we demonstrated that co‐exposure to combined Nic and NNK at SHS doses is more potent in inducing breast cell carcinogenesis than single‐agent exposures. We used our previously developed in vitro chronic‐induction carcinogenesis model . This model provides a highly appropriate approach and can use various endpoints (transient or constitutive) to study the effects of chronic exposure to environmental carcinogens on the development of sporadic breast cancer, in addition, using non‐tumorigenic breast cell line for chronic induction carcinogenesis considers as a good model for tracking the carcinogenicity progression stages, at an early point or at constitutive end point.…”

Curcumin reverses tobacco smoke‑induced epithelial‑mesenchymal transition by suppressing the MAPK pathway in the lungs of mice

“…EMT serves an important role in cancer initiation and development. The exposure of cells to carcinogens has been reported to induce EMT during transformation and tumor formation (35)(36)(37)(38)(39), indicating that EMT, by promoting cell malignancy transformation, may be associated with the initiation of tumorigenesis. The results of the present study were consistent with these reports, as they demonstrated that, following exposure to tobacco smoke for 12 weeks, tobacco alterations in EMT were observed in the lungs of mice.…”