Association of the Cytotoxic Effects of Waterpipe and Cigarette Smoke Extract with Epigenetic Changes in Breast Cancer Cells

Abstract

Background Smoking is one of the preventable leading causes of diseases and premature death worldwide. In Lebanon, smoking is a main public concern as it scored one of the highest smoking rate in the Eastern Mediterranean region reaching a peak of 53.9%. It is a major risk factor for the development of cancer including breast cancer, as according to the WHO in 2020, the incidence of breast cancer new cases ranked number one and became the second leading cause of death in Lebanon. Studies have shown a positive correlation between early and long term exposure to smoking and the incidence of breast cancer. Tobacco smoke was found to be behind the development of epigenetic aberrations that are linked to breast cancer, with the most compelling evidence for AhRR hypomethylation and cigarette smoking.

Aim The aim of this study is to determine the genotoxic and possible development of mesenchymal properties upon exposure to cytotoxic concentrations of cigarette and waterpipe smoke in MCF-7 and MDAMB-231 breast cancer cell lines, and to evaluate the differential methylation of the AhRR and MYT1L regions of interest that were found in peripherial blood samples to be specific for cigarette and waterpipe smoke respectively, in breast cancer cell lines.

Methods MTT assay was first done to determine the IC20 and IC50 for both waterpipe and cigarette smoke, and the concentrations were validated by trypan blue assay. These were then used to determine the genotoxic and cell cycle arrest effects using the ɣH2AX and flow cytometry assays respectively, and to determine any potential carcinogenic effect by evaluating the expression of epithelial and mesenchymal markers using the RT-PCR assay, and performing migration assay. Differential methylation of the AhRR and MYT1L regions of interest were then analyzed through direct bisulfide sequencing.

Results Exposure to cytotoxic concentrations of waterpipe and cigarette smoke caused DNA damage in MCF-7 and MDAMB-231 with secondary arrest at the S phase; though with the MDAMB-231 cell line exposure to higher concentrations showed less genotoxic damage that was translated into avoiding cell arrest at S phase. Also for both cell lines, the mesenchymal SNAIL marker increased with a trend of decrease in the CDH-1, an epithelial marker, but the results of the migration assay showed a decrease in migration ability of cells compared to the control at 24hrs. Finally, the differential methylation of the AhRR and MYT1L region of interests that were specific for peripheral blood samples were not so for breast cancer cell lines.

Conclusion Exposure to cytotoxic concentrations of cigarette and waterpipe smoke caused DNA damage and S phase cell cycle arrest. It also induced an increase in the expression of the SNAIL mesenchymal marker. The mesenchymal phenotype was observed under the microscope yet, and at high concentrations, MDAMB-231 cells may have become resistant to genotoxicity hence the highest expression of SNAIL. The lesser migration ability at 24hrs can be due to cell death. With respect to the epigenetic changes, results showed no difference in DNA methylation between the exposure and the control and the outcome was different from that of the peripheral blood samples.