DNA Methylation Changes Associated with Acute and Chronic Exposure of Waterpipe and Cigarette Smoke in A549 Cell Line

Abstract

Introduction: The tobacco epidemic is one of the biggest public health threats worldwide. Epidemiological studies showed that tobacco smoking increases the risk of developing several types of cancer, including lung cancer. Epigenetic changes, specifically DNA methylation, have been implicated in the development and progression of cancer. This study aimed to investigate the effects of acute (24h) and chronic (12 weeks) exposure to waterpipe (WPE) and cigarette (CSE) extracts on the phenotypic and DNA methylation changes in the A549 lung cancer cell line. Methods: MTT assay was used to determine the concentrations of WPE and CSE that caused 20% and 50% cell cytotoxicity (IC20 and IC50, respectively) in A549 cells. These concentrations were then validated using trypan blue assay. To evaluate the effect of acute exposure, A549 cells were then exposed to IC20 and IC50 concentrations of WPE and CSE for 24 hours. Then, migration ability was evaluated using scratch assay, while genotoxicity, cell cycle, and apoptosis were analyzed using flow cytometry. Additionally, the expression of epithelial to mesenchymal transition markers (EMT) was assessed using RT-qPCR. To evaluate the effect of chronic exposure, the cells were exposed to IC20 concentrations of WPE and CSE for 12 weeks. Expression of EMT markers and migration ability were assessed. In addition, DNA methylation changes in A549 cells after acute and chronic WPE and CSE exposure will be evaluated using whole-methylome analysis. Results: After 24 hours of exposure to WPE and CSE at either IC20 or IC50 concentrations, there was no statistically significant DNA damage, and no significant differences in cell migration distance compared to control. Moreover, there was no significant early or late apoptosis in the A549 cell line after exposure to WPE at both IC20 and IC50, while a statistically significant early apoptosis was observed with CSE at IC50. Similarly, there were statistically significant differences in % distribution of A549 cells in the SubG0 phase after WPE and CSE exposure at IC50, when compared to control. Yet, no statistically significant cell cycle arrest was observed for both WPE and CSE. Also, the expression of the epithelial marker E-cadherin decreased, while the expression of cytokeratin-7 (CK7) increased significantly. On the other hand, there was a significant decrease in the expression of the mesenchymal marker B-Catenin after WPE exposure at IC20.Yet, there was no change in the expression of neither epithelial markers nor in the mesenchymal markers after CSE exposure at IC20. As for chronic exposure after 12 weeks of exposure to WPE and CSE at IC20, there was a statistically significant increase in the distance travelled compared to control (at 12 and 24 hours of A549 cells) with CSE exposure only. Moreover, the expression of the mesenchymal markers SNAIL 1, SNAIL 2 and B-Catenin increased significantly after WPE and CSE exposure, in addition to an increase in Vimentin expression after CSE exposure only. DNA methylation after acute and chronic exposure are ongoing. Conclusions: This study showed that acute exposure of A549 cells to WPE and CSE did not cause DNA damage or affect cell migration ability. It also did not induce apoptosis except with exposure to CSE IC50 concentrations that caused statistically significant increase in early apoptosis. Moreover, IC50 exposure of both WPE and CSE caused cell death, as evidenced by the significant increase in the SubG0 phase, although there was no significant cell cycle arrest observed. Moreover, we observed a minimal but significant EMT transition after acute WPE exposure, with some contradictory results. However, no EMT transition was observed after CSE exposure. In contrast, chronic exposure of these same cells to CSE increased their migration ability, which was not the case for WPE. Furthermore, an EMT transition was observed after both WPE and CSE exposure. These findings suggest that prolonged exposure to both WPE and CSE may play a role in activating EMT signaling pathways and promoting the acquisition of a mesenchymal phenotype in A549 cells. We hope to decipher epigenetically deregulated biological pathways that may underlie this smoking-induced pathogenesis.