Abstract
Tobacco smoking has shown to change DNA methylation patterns, lead to the development and/or progression of oral cancer and cause alterations in transforming growth factor (TGF)-β signalling.The underlying hypothesis of this thesis is that change in the DNA methylation status of genes associated with TGF-β signalling in oral cells is a fundamental pathogenic mechanism by which tobacco smoke causes oral cancer.
The initial step was to investigate the expression of TGF-β protein in gingival tissue specimens from smokers and non-smokers and in oral squamous cell carcinoma (OSCC) using immunohistochemistry. The intensity of TGF-β staining was greatest for OSCC specimens, followed by that in smokers and least in non-smokers. A distinct and intense TGF-β expression was present in the OSCC specimens, which was used as a positive control in this study. The semi-quantitative analysis based on the integrated density of TGF-β staining found the smoker group to have a significantly higher immunoreactive score compared to non-smokers. The total number of TGF-β positive cells in each of the non-smoker, smoker and OSCC specimens was also counted, wherein a highly significant difference was found between the OSCC and non-smoker groups using a Kruskal-Wallis test followed by Dunn’s multiple comparisons. These findings imply that the data provides sufficient evidence to conclude that this model is plausible, yet it seems to be an over-simplified hypothesis since dealing with the complexities of OSCC pathogenesis, TGF-β signalling and mechanisms of tobacco-induced oral carcinogenesis.
This was followed by a series of in vitro experiments to comprehensively interrogate the effect of tobacco smoke on TGF-β signalling in human gingival fibroblasts (HGF) and oral epithelial cells (OEC) that may potentially lead to carcinogenesis. The cell culture experiments involved three cell lines each of primary HGF (HGF-1, HGF-2, HGF-3); commercial OEC (OKF-4, OKF-6, OKP-7) and OSCC (SCC-4, SCC-15, SCC-25). Commercial cigarette smoke condensate (CSC) was used to mimic tobacco smoking in vitro. Cellular viability of HGF and OEC was assessed across a range of CSC concentrations (0-600 µg/mL), their corresponding vehicle controls and time points (0-72hrs). Two-way ANOVA with post-hoc Dunnett’s multiple comparison test was used to compare the means for each CSC concentration and time points. HGF viability was found stimulated by lower CSC concentrations (50-100 µg/mL) while concentrations ≥200 µg/mL CSC were toxic. In OEC, there was a dose- and time-dependent decline in cellular viability with concentrations ≥50 µg/mL CSC being toxic to the cells at all time points.
Based on the above results, DNA and RNA was extracted from HGF treated with 100 and 200 µg/mL CSC and OEC treated with 25 and 50 µg/mL CSC for 72 hours, their untreated controls and from the three OSCC cell lines for molecular experiments. Foremost, tobacco smoke exposure was verified via gene expression studies to determine whether the in vitro findings could be applied in vivo. This was achieved by measuring Cyp1B1 mRNA levels in the CSC-treated HGFs, OECs and their untreated controls using quantitative real-time polymerase chain reaction (qPCR). Cyp1B1 is a key enzyme involved in the metabolism of tobacco carcinogens and is found upregulated in several tissues following tobacco exposure. Cyp1B1 gene expression was found affected by CSC treatment in both HGF and OEC, validating the in vitro tobacco smoke treatment model.
Thereafter, DNA methylation status of 22 candidate genes in the TGF-β signalling pathway was analysed using the EpiTect Methyl-Profiler DNA Methylation PCR Array System (Qiagen). This analysis was performed on DNA extracted from HGFs treated with 100μg/mL CSC and OECs treated with 50μg/mL CSC for 72 hours, along with their untreated controls. SMAD1 was found significantly hypermethylated following CSC treatment in HGF-1, while in OEC there was a significant CSC-induced hypermethylation of SMAD3 in OKF-4 and hypomethylation of TGFBR1 and LTBP2 in OKP-7. Methylation status of the three OSCC cell lines was also evaluated across the same panel of genes to assess if any changes caused by CSC in OEC were similar to those found in OSCC. Therein, genes BMP3 and GDF6 were significantly hypermethylated and genes BMP4, LTBP2, SMAD1, SMAD3 and TGFBR1 were found significantly hypomethylated compared with two or more OEC.
Based on the findings of this study, it is evident that CSC treatment causes DNA methylation changes in candidate genes of the TGF-β signalling pathway in both HGF and OEC. Given the small sample size and small methylation changes encountered, a link to DNA methylation patterns of the same genes in OSCC could not be established. Nonetheless, this study brought to light differential methylation in several novel genes that have not been reported vis-à-vis tobacco exposure and/or OSCC.