Areca nut alkaloids induce irreparable DNA damage and senescence in fibroblasts and may create a favourable environment for tumour progression.

BACKGROUND: Oral submucous fibrosis (OSMF) is a pre-malignant condition that is strongly associated with the areca nut alkaloids, arecoline (ARC) and arecaidine (ARD). The condition is characterised by the presence of senescent fibroblasts in the subepithelial mesenchyme which have the potential to promote malignancy in the neighbouring epithelial cells. We tested the hypothesis that areca nut alkaloids induce senescence in oral fibroblasts and promote the secretion of invasion-promoting transforming growth factor ß (TGF-ß) and matrix metalloproteinase-2 (MMP-2). METHODS: Two oral fibroblast lines were treated for 48h with ARC and ARD. Senescence-associated ß-galactosidase (SA-ßGal) activity, Ki67 (cycling cells), large 53BP1 foci (irreparable DNA strand breaks) and p16(INK) (4A) (late senescence) were used as markers of cellular senescence and were quantified using indirect immunofluorescence and the ImageJ program. TGF-ß and MMP-2 levels were measured using ELISA. Statistical analyses were performed with the two-tailed unpaired t-test where n = 3 and the Wilcoxon-Mann-Whitney test where n = 6. RESULTS: ARC (100 and 300 µM) and ARD (30 and 100 µM) significantly (P < 0.05) induced fibroblast senescence, as determined by the increased expression of SA-ßGal, 53BP1 staining and CDKN2A/p16(INK) (4A) ; there was also a non-significant reduction in Ki67 staining. Treated cells also showed a three- fivefold increase in TGF-ß and a small non-significant increase in MMP-2. CONCLUSIONS: Areca nut alkaloids induce senescence in oral fibroblasts and promote increased secretion of TGF-ß and perhaps MMP-2 that may create a tissue environment thought to be critical in the progression of OSMF to malignancy.

Senescent mesenchymal cells accumulate in human fibrosis by a telomere-independent mechanism and ameliorate fibrosis through matrix metalloproteinases.

Fibrosis can occur in many organs, where it is a debilitating and preneoplastic condition. The senescence of activated fibroblasts has been proposed to ameliorate fibrosis via the innate immune system but its role in humans has not been investigated. The availability of oral submucous fibrosis (OSMF) biopsies at different stages of disease progression allowed us to test the hypothesis that senescent fibroblasts accumulate with the progression of human fibrosis in vivo, and also to examine the mechanism of senescence. We tested the hypothesis that senescent cells may ameliorate fibrosis by increasing the secretion of matrix metalloproteinases (MMPs). We have used a combination of in situ immunodetection techniques, drug treatments, fluorescence-activated cell sorting and enzyme-linked absorbance assays on tissue samples and fibroblast cultures. We report a novel panning technique, based on fibronectin adhesion rates, to enrich and deplete senescent cells from fibroblast populations. Senescent fibroblasts, as determined by the presence of senescence-associated heterochromatic foci, accumulated with OSMF progression (R(2) = 0.98) and possessed a reduced replicative lifespan in vitro. Unlike wounds, however, OSMF fibroblasts were quiescent in vivo and consistent with this observation, possessed functional telomeres of normal length. Senescence was associated in vivo and in vitro with oxidative damage, DNA damage foci and p16(INK4A) accumulation and required the production of reactive oxygen species (ROS), perhaps from damaged mitochondria, but not the continuous presence of the disease stimulus (areca nut and tobacco), the tissue environment or other cell types. Depletion of OSMF fibroblasts of senescent cells showed that these cells accounted for 25-83 times more MMP-1 and -2 than their pre-senescent counterparts. The results show that the accumulation of senescent fibroblasts in human fibrosis occurs by a telomere-independent mechanism involving ROS and may locally ameliorate the condition by the increased expression of MMPs prior to clearance by the immune system.