Molecular alterations elicited by chronic exposure to cigarette smoke and chewing tobacco in oral keratinocytes
Tobacco is mainly consumed in two different forms (smoking and chewing) that vary in their composition and methods of intake. Despite being the leading cause of oral cancer, the molecular mechanisms resulting in malignancy upon tobacco exposure are yet to be fully elucidated. We therefore sought to compare the molecular alterations in oral keratinocytes exposed to smoke and chewing tobacco. OKF6/TERT1 cells were exposed to cigarette smoke condensate or chewing tobacco for progressively increasing durations (2, 4, 6 and 8 months). We employed a TMT-based quantitative proteomics approach to investigate the adverse effects of chronic cigarette smoke or chewing tobacco exposure in oral keratinocytes. LC/MS3 analysis resulted in the quantification of 5,342 proteins and 2,821 proteins in cigarette smoke and chewing tobacco exposed cells, respectively. Upstream regulator analysis indicates the involvement of distinct regulators in CSC exposed cells compared to STE exposed cells. In addition, exome sequencing revealed discrete genetic alterations in cells exposed to each insult. Current analysis defines a clear distinction in the molecular dysregulation in oral cells in response to different tobacco-based insults. Some of the proteins dysregulated in cigarette smoke or chewing tobacco exposed cells may serve as potential early detection biomarkers which could aid in stratification of patients based on tobacco usage history.
Sample Processing Protocol
OKF6-parental, OKF6-Tobacco (2-4-6-8M) and OKF6-Smoke (2-4-6-8M) cells were grown to 80% confluence and serum starved for 8h. Post-starvation, cells were washed with 1X PBS thrice and harvested in lysis buffer (2% SDS, 5 mM sodium fluoride, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate in 50 mM TEABC). The cell lysates were sonicated and centrifuged and protein concentration was determined by BCA. In-solution trypsin digestion of samples from both conditions was carried out. Equal amounts of cell lysate from all conditions were reduced using 5mM dithiothreitol (DTT) and incubated at 60°C for 20 mins. The reduced protein lysate was alkylated using IAA (20mM) and incubated for 10 mins in the dark at room temperature. The reduced and alkylated proteins were then precipitated using chilled acetone (1:5 v/v of sample) and incubation at -80oC for 4-6h to ensure complete removal of SDS from sample. The precipitated proteins were then subjected to trypsin digestion using sequencing grade trypsin (Promega, Madison, WI) at an enzyme to substrate ratio of 1:20. Trypsin digestion was carried out at 37°C for 12 – 16h. The digested peptides were labeled with TMT reagents as per manufacturers’ instructions. Briefly, peptide samples were dissolved in 50mM TEABC (pH 8.0) and added to TMT reagents dissolved in anhydrous acetonitrile. Peptides from OKF6-parental, OKF6-Smoke (2-4-6-8M) and OKF6-Tobacco (2-4-6-8M) were labeled with TMT tags as detailed below – A. OKF6-CSC temporal experiment – Condition TMT label Parental 128N CSC 2months (2M) 128C CSC 4months (4M) 129N CSC 6months (6M) 129C CSC 8months (8M) 130N B. OKF6-STE temporal experiment – Condition TMT label Parental 126 STE 2months (2M) 127C STE 4months (4M) 128C STE 6months (6M) 129C STE 8months (8M) 130C After incubation at room temperature for 1h, the reaction was quenched with 5% hydroxylamine. The labeled samples from all CSC exposed or STE exposed cells were pooled in two separate experiments and subjected to fractionation. LC-MS3 analysis of 12 concatenated bRPLC fractions for each separate experiment was performed in triplicates on Thermo Scientific™ Orbitrap Fusion™ Tribrid™ Mass Spectrometer.
Data Processing Protocol
Mass spectrometry data obtained from all LC-MS analysis were searched against Human RefSeq75 database (containing 36,690 entries along with common contaminants) using Sequest and Mascot (version 2.4.1) search algorithms through Proteome Discoverer 18.104.22.168 (Thermo Scientific, Bremen, Germany). Enzyme specificity was set as trypsin with maximum two missed cleavages allowed. The minimum peptide length was specified to be 6 amino acids. Carbamidomethylation of cysteine and TMT modification at N-terminus of the peptide and at lysine (K) were specified as fixed modifications and oxidation of methionine was included as variable modification. The mass error of parent ions was set to 10 ppm and 0.05 Da for fragment ions. The data was also searched against a decoy database and MS/MS identifications of < 1% false discovery rate (FDR) score threshold was considered for further analysis.
Aditi Chatterjee, Institute of Bioinformatics, Bangalore, Karnataka, India
Aditi Chatterjee, Institute of Bioinformatics, Unit 1, 7th floor, Discoverer building, ITPL, Whitefield, Bangalore - 560066, Karnataka, India ( lab head )
Rajagopalan P, Patel K, Jain AP, Nanjappa V, Datta KK, Subbannayya T, Mangalaparthi KK, Kumari A, Manoharan M, Coral K, Murugan S, Nair B, Prasad TSK, Mathur PP, Gupta R, Gupta R, Khanna-Gupta A, Califano J, Sidransky D, Gowda H, Chatterjee A. Molecular alterations associated with chronic exposure to cigarette smoke and chewing tobacco in normal oral keratinocytes. Cancer Biol Ther. 2018 19(9):773-785 PubMed: 29723088