Increased senescence and altered ECM remodelling in oral submucous fibrosis

Background: Oral submucous fibrosis (OSMF) is a pre-neoplastic condition, causally linked to areca nut consumption, the pathogenesis of which is poorly understood. TGF-β and disturbances in the balance between MMPs and TIMPs have been implicated in increased collagen deposition and fibrosis but no previous study has addressed the role of mesenchymal senescence in OSMF. Materials ;Methods: Senescence and its secretome, DNA damage, oxidative damage, ROS production and mitochondrial damage were studied in OSMF in vivo and in vitro using ELISA, immunofluorescence, western blot and FACS techniques. Results: Senescent cells increased in all OSMF samples (1.9±0.3; p=0.004) peaking when dysplasia was present in the OSMF epithelium. There was increased oxidative damage (6.7±1.8; p=0.004); elevated DSBs (10.4±1.1; p=0.004) and P16ink4a accumulation (4.2±1.7; p=0.004). The results were similar in vitro. The OSMF fibroblasts demonstrated a reduced replicative lifespan (MPD-22±7.2; p=0.0001), despite having normal telomere lengths and in vivo growth rates. However, the OSMF fibroblasts showed increased ROS production and increased mitochondrial density and hyperpolarization, suggesting mitochondrial damage. The antioxidant, N-tert-Butyl-α- phenylnitrone (PBN) reduced the frequency of senescent fibroblasts and their associated markers in both OSMF and the control cultures. The mild uncoupling of mitochondrial oxidative phosphorylation from ROS generation with dinitrophenol (DNP) gave similar results. Cytokine profiles from cells obtained from OSMF showed a significant elevation of TIMP-1 (2217.4±406.5; p=0.003) and TIMP-2 (1763.2+/-363.7 pg/ml; p=0.004) production as compared to normal. The levels of MMP-1 (7.0±2.2 ng/ml; p=0.30), MMP-2 (157.8±91.3 ng/ml; p=0.75) and TGF-β1 (389.5±250.6 ng/ml; p=0.22) were not different to the normal and non-diseased controls (ND) collagen production was not elevated in OSMF in vitro (20.6±4.4 μg/ml; p=0.22). 3 Conclusion: Senescence, DNA damage, oxidative damage and P16inka accumulation are associated with neoplastic progression of OSMF. Elevated TIMP levels did not result in increased collagen secretion but may be an early marker of fibroblast aging and senescence.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Clinical and histopathologic parameters in survival of oral squamous cell carcinoma

Objective. The aim of this study was to assess the relevance of clinical and histopathologic parameters on survival of oral squamous cell carcinoma (OSCC) patients in Sri Lanka. Study Design. A cohort of 193 previously diagnosed OSCC patients were followed for up to 5 years. Clinical and histopathologic parameters were analyzed regarding local recurrence and 5-year survival after treatment. Results. Site, stage, local recurrence, degree of differentiation, degree of keratinization, pattern of invasion, and status of the excision margins showed a significant association with the 5-year survival (P<.05). Multivariate analysis identified stage, pattern of invasion, and status of the excision margins as true independent prognostic indicators of OSCC. Pattern of invasion was the best prognosticator of both local recurrence and overall survival in the Cox regression model (P<.001). Conclusions. Stage, pattern of invasion, and status of the excision margins are superior prognostic indicators of OSCC compared with other parameters. © 2012 Elsevier Inc. All rights reserved.Link_to_subscribed_fulltex

Progression of genotype-specific oral cancer leads to senescience of cancer associated fibroblasts and is mediated by oxidative stress and TGF-B

Keratinocyte senescence acts as a barrier to tumor progression but appears to be lost in late pre-malignancy to yield genetically unstable oral squamous cell carcinomas (GU-OSCC); a subset of OSCC possessing wild-type p53 and are genetically stable (GS-OSCC). In this study, fibroblasts from GU-OSCC were senescent relative to fibroblasts from GS-OSCC, epithelial dysplastic tissues or normal oral mucosa, as demonstrated by increased senescence-associated ?-galactosidase (SA ?-Gal) activity and overexpression of p16(INK4A). Keratinocytes from GU-OSCC produced high levels of reactive oxygen species (ROS) and this was associated with an increase in the production of transforming growth factor-?1 (TGF-?1) and TGF-?2 in stromal fibroblasts. Treatment of normal fibroblasts with keratinocyte conditioned media (CM) from GU-OSCC, but not GS-OSCC or dysplastic keratinocytes with dysfunctional p53, induced fibroblast senescence. This phenomenon was inhibited by antioxidants and anti-TGF-? antibodies. Fibroblast activation by TGF-?1 preceded cellular senescence and was associated with increased ROS levels; antioxidants inhibited this reaction. Senescent fibroblasts derived from GU-OSCC or normal fibroblasts treated with CM from GU-OSCC or hydrogen peroxide, but not non-senescent fibroblasts derived from GS-OSCC, promoted invasion of keratinocytes in vitro. Epithelial invasion was stimulated by fibroblast activation and amplified further by fibroblast senescence. The data demonstrate that malignant keratinocytes from GU-OSCC, but not their pre-malignant counterparts, produce high levels of ROS, which, in turn, increase TGF-?1 expression and induce fibroblast activation and senescence in a p5-independent manner. Fibroblasts from GU-OSCC were particularly susceptible to oxidative DNA damage because of high levels of ROS production, downregulation of antioxidant genes and upregulation of pro-oxidant genes. The results demonstrate the functional diversity of cancer-associated fibroblasts and show that malignant keratinocytes from GU-OSCC reinforce their malignant behavior by inducing fibroblast activation and senescence through ROS and TGF-?-dependent mechanism

Senescent Human Fibroblasts Show Increased Glycolysis and Redox Homeostasis with Extracellular Metabolomes That Overlap with Those of Irreparable DNA Damage, Aging, and Disease

Cellular senescence can modulate various pathologies and is associated with irreparable DNA double-strand breaks (IrrDSBs). Extracellular senescence metabolomes (ESMs) were generated from fibroblasts rendered senescent by proliferative exhaustion (PEsen) or 20 Gy of γ rays (IrrDSBsen) and compared with those of young proliferating cells, confluent cells, quiescent cells, and cells exposed to repairable levels of DNA damage to identify novel noninvasive markers of senescent cells. ESMs of PEsen and IrrDSBsen overlapped and showed increased levels of citrate, molecules involved in oxidative stress, a sterol, monohydroxylipids, tryptophan metabolism, phospholipid, and nucleotide catabolism, as well as reduced levels of dipeptides containing branched chain amino acids. The ESM overlaps with the aging and disease body fluid metabolomes, supporting their utility in the noninvasive detection of human senescent cells in vivo and by implication the detection of a variety of human pathologies. Intracellular metabolites of senescent cells showed a relative increase in glycolysis, gluconeogenesis, the pentose-phosphate pathway, and, consistent with this, pyruvate dehydrogenase kinase transcripts. In contrast, tricarboxylic acid cycle enzyme transcript levels were unchanged and their metabolites were depleted. These results are surprising because glycolysis antagonizes senescence entry but are consistent with established senescent cells entering a state of low oxidative stress