Fibroblasts May Induce Epithelial Hyperplasia in Users of Smokeless Tobacco

Epithelial hyperplasia is a characteristic of smokeless tobacco(ST)-induced changes in oral mucosa. Keratinocyte growth factor KGF and hepatocyte growth factor HGF and granulocyte/macrophage colony stimulating factor GM-CSF are fibroblast-derived growth factors with mitogenic effects on epithelial cells. The Objectives: To investigate the effect of ST on 1) HGF, KGF and GM-CSF expression by buccal fibroblasts 2) on keratinocyte and fibroblast proliferation. Methods: Buccal fibroblasts were grown to confluence and stimulated with different concentrations of ST extracts in a double dilution from 0.5% w/v to 0.03% w/v. Supernatant was collected after 24/ 48 /72 /96 h and assayed for HGF, KGF and GM-CSF by ELISA. The amount of RNA was determined by Ribogreen® assay as an indicator of fibroblast cell number. Buccal epithelial cell proliferation was determined by CyQUANT® proliferation assay in primary cultures with culture medium 0.25% w/v and 0.5% w/v ST. Results: The amount of HGF in the supernatant was dependent on time and concentration of the tobacco extract. High concentration increased production of HGF 4 fold whereas low concentration lead to 2-fold increase at 72 h. KGF production was doubled when high concentration of tobacco was used, low concentration showed no difference from non-stimulated cells. GM-CSF production was low in both stimulated and non-stimulated cells, the level being at a detection level in all experiments. Keratinocytes and fibroblasts showed no increase in proliferation after stimulation with increased concentrations of T. Conclusion: The results suggest that HGF and KGF may play an important role as a paracrine growth factor in epithelial hyperplasia in ST lesions.