THE EPIGENETIC CHANGES ASSOCIATED WITH ACUTE AND CHRONIC EXPOSURE TO CIGARETTE AND WATERPIPE SMOKE EXTRACTS ON A COLON CANCER CELL LINE

Abstract

Introduction Tobacco smoking is a major public health issue worldwide, and it has been linked to various health risks, including colon cancer. While the link between cigarette smoking and colon cancer is well established, the extent to which waterpipe smoking causes phenotypic and molecular changes in colon cancer requires further investigation. Both cigarette and waterpipe smoking may promote colon cancer development and progression through different mechanisms, including epigenetic modulation, such as DNA methylation. The aim of this study is to identify the phenotypic effects and the underlying epigenetic mechanisms through which cigarette smoke (CSE) and waterpipe smoke (WPE) extracts affect the HCT116 colon cancer cell line.

Methods MTT assay was initially conducted to determine the inhibitory concentrations leading to 20% and 50% cell cytotoxicity (IC20 and IC50, respectively) for both CSE and WPE. Subsequently, these concentrations were validated using the trypan blue assay. The cell line was then acutely exposed to IC20 and IC50 of CSE and WPE for 24 hours to study the genotoxic, cell cycle, and apoptosis phenotypic effects, using γH2Ax, PI, and annexin/PI staining assays, respectively, acquired by flow cytometry, as well as RNA expression of epithelial to mesenchymal transition (EMT) markers by RT-qPCR. Furthermore, scratch assay was conducted to investigate the effects of acute exposure on the migration process, but only with IC20. In addition, the HCT116 cells were chronically exposed to IC20 of CSE and WPE over 12 weeks followed by migration and EMT analysis. Whole methylome analysis will be conducted to assess alterations in DNA methylation upon acute and chronic exposures.

Results Exposure to IC50 of CSE and WPE for 24 hours induced statistically significant genotoxic effects and apoptosis in HCT116 cells. However, there were no statistically significant differences in cell cycle and migration assays following acute exposure to both extracts, when compared to control. In the migration assay after chronic exposure, a decrease in the migration ability was observed with WPE, while an increase was observed with CSE. Analysis of EMT markers revealed that acute exposure to IC20 of WPE and CSE resulted in an increase in the mesenchymal markers SNAIL 1 and SNAIL2, along with a slight increase in CK7 epithelial marker. Chronic exposures resulted in a significant slight decrease in the CK7 epithelial marker and SNAIL 1 mesenchymal marker, and an evident increase in the mesenchymal marker Vimentin. Notably, whole Genome-wide DNA methylation analysis is currently in progress and its results are pending.

Conclusion Our results indicate that CSE and WPE have pro-apoptotic and genotoxic effects on HCT116 cells. These effects are only observed at higher concentrations (IC50) and not at sub-cytotoxic concentrations (IC20). Moreover, both extracts appear to trigger the acquisition of mesenchymal characteristics in the cells. Furthermore, chronic exposure to IC20 of WPE and CSE showed differential effects on cell migration after 24hours.We hope to decipher epigenetically deregulated biological pathways that may underlie this smoking-induced progression.