Project description:Cigarette smoking exerts diverse physiological effects including immune suppression. Existing US epidemiological data show that consumption of smokeless tobacco products, such as moist snuff, is less harmful relative to cigarette smoking. In efforts to understand the molecular changes due to consumption of different tobacco product classes, we have shown recently that smokers exhibit distinct peripheral blood mononuclear cells (PBMCs) gene expression patterns relative to moist snuff users and non-tobacco consumers (NTCs). To better characterize the biological effects exerted from the use of different tobacco products, a genome-wide gene expression study, using a cultured PBMC model, was performed. Global gene expression profiling results showed that 5,421 genes (2,809 upregulated and 2,612 downregulated) were dose-dependently changed by WSCM (pFDR < 0.01). Some gene expression changes detected in the cultured system were also observed in clinical studies. For example, FEZ1 and SLAMF7 were suppressed by WSCM while CCR2 and HHEX genes were upregulated by WSCM commonly in the cultured experiment and clinical studies. WSCM exposures, but not STE, uniquely affected genes involved in immune cell development and inflammatory response. Ingenuity Pathway Analysis identified upstream regulators, such as TNF, IL1β, and NFκB to be likely responsible for the observed gene expression changes and that these cascading signals were generally suppressed by WSCM, but not STE. Collectively, these findings sµggested that combustible and non-combustible tobacco products produce distinct biological effects which could explain the observed chronic immune suppression in smokers.
Project description:Tobacco in its smoke and smokeless form are major risk factors for ESCC (esophageal squamous cell carcinoma). However, molecular alterations associated with smokeless tobacco exposure are poorly understood. In the Indian subcontinent, tobacco is predominantly consumed in chewing form. An understanding of molecular alterations associated with chewing tobacco exposure is vital for identifying molecular markers and potential targets. We developed an in-vitro cellular model by exposing non-transformed esophageal epithelial cells to chewing tobacco over eight month period. Chronic exposure to chewing tobacco led to increase in cell proliferation, invasive ability and anchorage independent growth indicating cell transformation. Molecular alterations associated with chewing tobacco exposure were characterized by carrying out exome sequencing and quantitative proteomic profiling of parental cells and chewing tobacco exposed cells. Quantitative proteomic analysis revealed that established cancer stem cell markers are elevated in tobacco treated cells. Decreased expression of enzymes associated with the glycolytic pathway and increased expression of a large number of mitochondrially localized proteins involved in the electron transport chain as well as enzymes of TCA cycle were also identified. Electron micrographs revealed increase in number and size of mitochondria. Based on these observations, we hypothesise that chronic treatment of esophageal epithelial cells with tobacco leads to a cancer stem cell-like phenotype. These cells also show characteristic OXPHOS phenotype which can be potentially targeted as a therapeutic strategy.
Project description:The practice of using chewing tobacco is common in certain socio-economic sections of southern Asia particularly in the Indian subcontinent. The molecular mechanism of smokeless tobacco which leads to malignancy is unclear. Chewing tobacco demonstrates a carcinogenic effect through chronic and not acute exposures. Using a cell line model, we studied the chronic effects of chewing tobacco on the proteome in normal oral keratinocytes (OKF6/TERT1). We carried out iTRAQ-based quantitative proteomic analysis of the untreated and chewing tobacco treated oral keratinocytes. LC-MS/MS analysis of this cell line pair resulted in the identification of 3,638 proteins of which 408 were found to be differentially expressed.