Orren, DavidAlqithami, Sarah2025-07-142025-05-09https://hdl.handle.net/20.500.14154/75822Cigarette smoke is a complex mixture of over 7,000 chemicals, many of which are known carcinogens and pro-inflammatory agents that contribute to chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. This study aimed to investigate the cellular and molecular responses of human bronchial epithelial cells (HBECs) to cigarette smoke condensate (CSC), focusing on oxidative stress and the NF-κB signaling pathway. Three HBEC lines (HBEC3KT, HBEC14, and HBEC2) were exposed to CSC (20 µg/mL) for 48 hours, a dose shown to induce morphological changes consistent with an epithelial-to-mesenchymal transition (EMT) phenotype without causing overt cytotoxicity. RNA sequencing revealed significant differential gene expression in all three cell lines upon CSC exposure. Principal component analysis (PCA) and hierarchical clustering demonstrated clear transcriptomic shifts, particularly in genes related to oxidative stress (e.g., NQO1, GCLM, HMOX1), inflammatory responses (e.g., IL6, CXCL8, TNFAIP3), hypoxia (e.g., CA9, VEGFA, HIF1A), and metabolic regulation. Enrichment analyses further confirmed activation of the NRF2 antioxidant pathway, pro-inflammatory NF-κB and IL-17 signaling, and hypoxia-inducible factor (HIF) pathways, indicating a coordinated stress response to CSC. Western blot and immunofluorescence analyses supported transcriptomic findings by demonstrating increased protein expression of phosphorylated NF-κB p65 in CSC-treated cells, particularly in HBEC3KT, confirming its activation in response to smoke-induced EMT. Scratch wound healing assays showed that CSC treatment enhanced the migratory ability of HBEC3KT cells, resulting in faster wound closure. These findings suggest that CSC may positively influence cell migration and tissue repair. Additionally, a decrease in the expression of E-cadherin (CDH1) and an increase in vimentin (VIM) and indicated partial epithelial-mesenchymal transition (EMT). These findings were corroborated by gene expression patterns and validated through protein-level analyses. Cell viability assays demonstrated moderate cytotoxicity following 48-hour CSC treatment, with HBEC2 being the most sensitive. These results suggest a potential state of chronic cellular stress, inflammation, and hypoxia, along with indications of impaired epithelial restitution, although these effects have not yet been directly demonstrated in lung tissue, all of which are hallmark features of early airway remodeling in smoke-related lung pathologies. In summary, this study comprehensively elucidates the transcriptional and functional responses of bronchial epithelial cells to cigarette smoke condensate. The integration of transcriptomic, proteomic, and functional data highlights the central roles of oxidative stress, inflammation, and epithelial dysfunction in smoke-induced airway injury. These findings provide mechanistic insights into early events driving airway remodeling and inflammation in smokers and may inform future therapeutic strategies targeting airway epithelial resilience.198en-USCigarette Smoke Condensate (CSC)Human Bronchial Epithelial Cells (HBECs)Oxidative StressNF-κB SignalingEpithelial-Mesenchymal Transition (EMT)Chronic Obstructive Pulmonary Disease (COPD)Thesis