Waterpipe smoke condensate influences epithelial to mesenchymal transition and interferes with the cytotoxic immune response in non-small cell lung cancer cell lines

Abstract

Waterpipe tobacco smoking (WPS) continues to spread globally and presents serious health hazards. The aim of the present study was to investigate the effects of treatment with WPS condensate (WPSC) on lung cell proliferation and plasticity as well as tumor cell recognition and killing by natural killer (NK) cells using cytotoxicity assays. The results indicated that exposure of normal and cancer lung cell lines to WPSC resulted in a decrease in their in vitro growth in a dose-dependent manner and it induced tumor senescence. In addition, WPSC selectively caused DNA damage as revealed by an increase in γH2AX and 53BP1 in tumor lung cells. To gain further insight into the molecular mechanisms altered by WPSC, we conducted a global comprehensive transcriptome analysis of WPSC-treated tumor cells. Data analysis identified an expression profile of genes that best distinguished treated and non-treated cells involving several pathways. Of these pathways, we focused on those involved in epithelial to mesenchymal transition (EMT) and stemness. Results showed that WPSC induced an increase in SNAI2 expression associated with EMT, ACTA2 and SERPINE2 were involved in invasion and CD44 was associated with stemness. Furthermore, WPSC exposure increased the expression of inflammatory response genes including CASP1, IL1B, IL6 and CCL2 . While immune synapse formation between NK and WPSC-treated lung cancer target cells was not affected, the capacity of NK cells to kill these target cells was reduced. The data reported in the present study are, to the best of our knowledge, the first in vitro demonstration of WPSC effects on lung cellular parameters providing evidence of its potential involvement in tumor physiology and development.

Genomic Analysis of Waterpipe Smoke-Induced Lung Tumor Autophagy and Plasticity

“…For this purpose, both A549 and H460 lung cancer cell lines were treated with 0.5% WPSC. This WPSC concentration was previously found to cause only a small fraction of A549 and H460 cells to die [ 11 ]. Cell viability using the MTT assay at 24, 48 and 72 h was measured.…”

“…Accumulated evidence indicates that smoke plays a central role in the evolution of tumor ecosystem and immune escape mechanisms by tumor cells through its impact on immune plasticity and tumor heterogeneity. In this regard, we had previously observed that treating lung cancer cell lines with WPSC resulted in an increase in DNA damage [ 11 ]. Here, we asked whether WPSC interferes with the autophagic process and how this may influence the immune landscape in the lung of smokers.…”

“…The cells that escape apoptosis can either undergo autophagy or senescence. While elevated levels of autophagy induce cell death, inadequate autophagy can trigger cellular senescence [ 38 ], which we have previously shown is also induced following 8-day treatment with the same concentrations of WPSC [ 11 ]. While DNA damage potentiates different repair mechanisms to restore the damaged DNA, which, if unrepaired, would lead to the activation of cell death programs [ 39 ], autophagy has been shown to function in delaying apoptotic cell death in cancers as autophagy inhibition sensitizes cancer cells to chemotherapeutic drugs and/or ionizing radiation [ 32 , 40 , 41 , 42 , 43 ] and is also shown to play a role in the inhibition of the immune response in cancers with high TMB [ 44 ].…”

“…Previous studies have shown that WPS condensate (WPSC) treatment of lung cancer cell lines modulates cell plasticity. WPSC induced epithelial to mesenchymal transition (EMT), cancer stem cell (CSC) features, and an increase in inflammation and DNA damage [ 11 , 12 ]. The consequences of DNA damage depend on the cell type and on the extent and intensity of the stress and could activate senescence, autophagy, or cell death programs.…”

Key challenges for in vitro testing of tobacco products for regulatory applications: Recommendations for dosimetry

“…73 Extracts from other products, including cigars or waterpipe, have been collected under suitable smoking regimes. [74][75][76][77] More recently, waterpipe tobacco has been smoked using the standardized ISO 22486 78 conditions. 79 A wide range of smoking or puffing machines have been used to generate whole smoke or aerosol for in vitro exposure systems.…”