DIFFERENT ROLES OF AHR IN LUNG ADENOCARCINOMA EPITHELIAL CELLS AFTER PROLONGED EXPOSURE TO TCDD AND BAP

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027162%3A_____%2F19%3AN0000253" target="_blank" >RIV/00027162:_____/19:N0000253 - isvavai.cz</a>

Result on the web

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DOI - Digital Object Identifier

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Result language

angličtina

Original language name

DIFFERENT ROLES OF AHR IN LUNG ADENOCARCINOMA EPITHELIAL CELLS AFTER PROLONGED EXPOSURE TO TCDD AND BAP

Original language description

DIOXIN 2019, Kyoto, 25. – 30.8.2019 – poster. Introduction: Environmental pollutants, e.g. components of cigarette smoke, herbicides and airborne particles, have been previously documented as potent inducers of AhR signaling in lung tissue. Although several reports provide indications that AhR signaling plays an important role in the progression and dissemination of lung primary tumors, the mechanism of this process remains rather elusive. Here, we employed a well-annotated in vitro model of lung carcinoma cells A549 and investigated their cellular fate after chronic exposure to model agonistic AhR ligands (benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)). Materials and methods: The A549 human cancer lung epithelial cells were exposed to 1 µM BaP, 10 nM TCDD or 2 ng/ml transforming growth factor -ß1 (TGF) as a model EMT inducer for 14 days. During the experiments the cells were replaced twice a week in medium with the tested compounds. Cell proliferation was measured by counting the cells with a Coulter Counter (Beckman) and cell cycle distribution was analyzed using by flow cytometer (FACSCalibur, Becton Dickinson). Cellular morphology was visualized by Alexa Fluor 488-conjugated phalloidin/DAPI staining following by examination on a Leica SP8 confocal scanning laser microscope. Bright field images were taken using Nikon TS1 100 phase-contrast microscope. Cell migration potential was analyzed using Corning® Transwell® polycarbonate membrane 24-well cell culture inserts (Sigma Aldrich). Levels of mRNA were determined by qRT-PCR and protein levels by Western blotting. Lentiviral delivery of hCDKN1A-shRNA into commercial pLKO vectors was used to create A549 cell clones with reduced expression of p21. Total RNA samples isolated from 2 week exposed A549 cells were handled according to the Illumina HumanHT-12 v4 Expression BeadChip manufacturer's instruction (Illumina Inc, San Diego, CA, USA). Functional analysis of microarray data was performed using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) software (Qiagen, Redwood, CA, USA). Results: We found significant differences in phenotypes of A549 cells after prolonged (14 days) exposure to TCDD and BaP. While TCDD slightly increased the proliferation of A549 cells, exposure to BaP was associated with the cell cycle arrest and inhibition of proliferation. Moreover, unlike TCDD exposure, A549 cells prolonged exposed to BaP switched to EMT-like phenotype. The transformation was associated with enhancement of migratory potential and with changes in morphology and expression pattern of EMT markers (E-cadherin, N-cadherin, Snail), and was observed similarly after prolonged exposure to TGF, a model inducer of EMT. Based on the results obtained from transcriptome analysis and due to the fact that BaP a TCDD had opposite effects on A549 cell proliferation, we suggested the possible role of cell cycle arrest in BaP-mediated induction of EMT-like phenotype in A549 cells. Thus, we next attempted to inhibit A549 cell growth using a low concentration of MMC (10 ng/ml). Prolonged exposure to both mitomycin and TCDD resulted in a shift of the A549 cells toward EMT-like phenotype. Since BaP as a genotoxic compound triggers cell cycle arrest by p53-mediated induction of p21 expression, we next generated the A549 cell clone with reduced p21 expression using hCDKN1A-shRNA and exposed them to BaP and TGF for 2 weeks. The reduction of p21 expression led to the significant suppression of EMT-like phenotype induction in A549 cells during the prolonged exposure to BaP, and surprisingly, to slight reduction in case of TGF exposure. Conclusion: Our findings support the hypothesis that cell cycle arrest associated with increased p21 expression and attenuation of A549 proliferation are essential for AhR-mediated induction of EMT-like phenotype in lung adenocarcinoma cells, and, thus, could play a role in AhR-mediated progression of lung primary tumors.

Czech name

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Czech description

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Type

O - Miscellaneous

CEP classification

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OECD FORD branch

10601 - Cell biology

Project

<a href="/en/project/GA19-25365S" target="_blank" >GA19-25365S: The mechanisms of carcinogenic processes in normal and transformed airway cellular models exposed to airborne aromatic toxicants</a><br>

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Publication year

2019

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů