Dyskeratosis Congenita Links Telomere Attrition to Age-Related Systemic Energetics.

The underlying mechanisms of plasma metabolite signatures of human aging and age-related diseases are not clear but telomere attrition and dysfunction are central to both. Dyskeratosis congenita (DC) is associated with mutations in the telomerase enzyme complex (TERT, TERC, and DKC1) and progressive telomere attrition. We analyzed the effect of telomere attrition on senescence-associated metabolites in fibroblast-conditioned media and DC patient plasma. Samples were analyzed by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. We showed extracellular citrate was repressed by canonical telomerase function in vitro and associated with DC leukocyte telomere attrition in vivo, leading to the hypothesis that altered citrate metabolism detects telomere dysfunction. However, elevated citrate and senescence factors only weakly distinguished DC patients from controls, whereas elevated levels of other tricarboxylic acid cycle (TCA) metabolites, lactate, and especially pyruvate distinguished them with high significance. The DC plasma signature most resembled that of patients with loss of function pyruvate dehydrogenase complex mutations and that of older subjects but significantly not those of type 2 diabetes, lactic acidosis, or elevated mitochondrial reactive oxygen species. Additionally, our data are consistent with further metabolism of citrate and lactate in the liver and kidneys. Citrate uptake in certain organs modulates age-related disease in mice and our data have similarities with age-related disease signatures in humans. Our results have implications for the role of telomere dysfunction in human aging in addition to its early diagnosis and the monitoring of anti-senescence therapeutics, especially those designed to improve telomere function.

Anticancer effects of putative and validated BH3-mimetic drugs in head and neck squamous cell carcinomas: An overview of current knowledge.

The purpose of this review was to summarise available literature concerning the anticancer effects of both putative and validated BH3-mimetics in head and neck squamous cell carcinomas. A literature search was performed and studies assessing malignant cell lines, xenograft models, and/or humans were considered eligible. A total of 501 studies were identified, of which 40 were included. One phase-II clinical trial assessing gossypol (combined with docetaxel) was found. The remaining 39 preclinical studies investigated cell lines and/or xenograft models involving the use of six validated BH3-mimetics (A-1210477, A-1331852, ABT-737, navitoclax, S63845, venetoclax) and six putative BH3-mimetics (ApoG2, gossypol, obatoclax, sabutoclax, TW-37, and YC137). In preclinical settings, most validated BH3-mimetics were capable of inducing apoptosis (in-vitro) and tumour growth inhibition (in-vivo). The majority of putative BH3-mimetics were also capable of inducing cell death, although important off-target effects, such as autophagy induction, were also described. Combinations with conventional anticancer drugs, ionising radiation, or multiple BH3-mimetics generally resulted in enhanced anticancer effects, such as increased sensitivity to apoptotic stimuli, especially considering some cell lines that showed resistance to either treatment alone. In conclusion, although clinical data are still insufficient to evaluate the anticancer effects of BH3-mimetics in head and neck squamous cell carcinomas, promising results in preclinical settings were observed concerning induction of cell death and inhibition of tumour growth. Therefore, further clinical trials are highly encouraged.

Extracellular Prostaglandins E1 and E2 and Inflammatory Cytokines Are Regulated by the Senescence Program in Potentially Premalignant Oral Keratinocytes.

Potentially pre-malignant oral lesions (PPOLs) are composed of keratinocytes that are either mortal (MPPOL) or immortal (IPPOL) in vitro. We report here that MPPOL, but not generally IPPOL, keratinocytes upregulate various extracellular tumor-promoting cytokines (interleukins 6 and 8) and prostaglandins E1 (ePGE1) and E2 (ePGE2) relative to normal oral keratinocytes (NOKs). ePGE upregulation in MPPOL was independent of PGE receptor status and was associated with some but not all markers of cellular senescence. Nevertheless, ePGE upregulation was dependent on the senescence program, cyclo-oxygenase 2 (COX2) and p38 mitogen-activated protein kinase and was partially regulated by hydrocortisone. Following senescence in the absence of p16INK4A, ePGEs accumulated in parallel with a subset of tumor promoting cytokine and metalloproteinase (MMP) transcripts, all of which were ablated by ectopic telomerase. Surprisingly, ataxia telangiectasia mutated (ATM) function was not required for ePGE upregulation and was increased in expression in IPPOL keratinocytes in line with its recently reported role in telomerase function. Only ePGE1 was dependent on p53 function, suggesting that ePGEs 1 and 2 are regulated differently in oral keratinocytes. We show here that ePGE2 stimulates IPPOL keratinocyte proliferation in vitro. Therefore, we propose that MPPOL keratinocytes promote the progression of IPPOL to oral SCC in a pre-cancerous field by supplying PGEs, interleukins and MMPs in a paracrine manner. Our results suggest that the therapeutic targeting of COX-2 might be enhanced by strategies that target keratinocyte senescence.

Metabolic Alterations in Cellular Senescence: The Role of Citrate in Ageing and Age-Related Disease.

Recent mouse model experiments support an instrumental role for senescent cells in age-related diseases and senescent cells may be causal to certain age-related pathologies. A strongly supported hypothesis is that extranuclear chromatin is recognized by the cyclic GMP-AMP synthase-stimulator of interferon genes pathway, which in turn leads to the induction of several inflammatory cytokines as part of the senescence-associated secretory phenotype. This sterile inflammation increases with chronological age and age-associated disease. More recently, several intracellular and extracellular metabolic changes have been described in senescent cells but it is not clear whether any of them have functional significance. In this review, we highlight the potential effect of dietary and age-related metabolites in the modulation of the senescent phenotype in addition to discussing how experimental conditions may influence senescent cell metabolism, especially that of energy regulation. Finally, as extracellular citrate accumulates following certain types of senescence, we focus on the recently reported role of extracellular citrate in aging and age-related pathologies. We propose that citrate may be an active component of the senescence-associated secretory phenotype and via its intake through the diet may even contribute to the cause of age-related disease.

Desmoglein-3 induces YAP phosphorylation and inactivation during collective migration of oral carcinoma cells.

Alterations of the Hippo-YAP pathway are potential targets for oral squamous cell carcinoma (OSCC) therapy, but heterogeneity in this pathway could be responsible for therapeutic resistance. We analysed the Hippo-YAP signatures in a cohort of characterised keratinocyte cell lines derived from the mouth floor and buccal mucosa from different stages of OSCC tumour progression and focused on the specific role of YAP on invasive and metastatic potential. We confirmed heterogeneity in the Hippo-YAP pathway in OSCC lines, including overexpression of YAP1, WWTR1 (often referred to as TAZ) and the major Hippo signalling components, as well as the variations in the genes encoding the intercellular anchoring junctional proteins, which could potentially regulate the Hippo pathway. Specifically, desmoglein-3 (DSG3) exhibited a unique and mutually exclusive regulation of YAP via YAP phosphorylation during the collective migration of OSCC cells. Mechanistically, such regulation was associated with inhibition of phosphorylation of epidermal growth factor receptor (EGFR) (S695/Y1086) and its downstream effectors heat shock protein beta-1 (Hsp27) (S78/S82) and transcription factor AP-1 (c-Jun) (S63), leading to YAP phosphorylation coupled with its cytoplasmic translocation and inactivation. Additionally, OSCC lines displayed distinct phenotypes of YAP dependency or a mixed YAP and TAZ dependency for cell migration and present distinct patterns in YAP abundance and activity, with the latter being associated with YAP nuclear localisation. In conclusion, this study provides evidence for a newly identified paradigm in the Hippo-YAP pathway and suggests a new regulation mechanism involved in the control of collective migration in OSCC cells.

The Extracellular Metabolome Stratifies Low and High Risk Potentially Premalignant Oral Keratinocytes and Identifies Citrate as a Potential Non-Invasive Marker of Tumour Progression.

Premalignant oral lesions (PPOLs) which bypass senescence (IPPOL) have a much greater probability of progressing to malignancy, but pre-cancerous fields also contain mortal PPOL keratinocytes (MPPOL) that possess tumour-promoting properties. To identify metabolites that could potentially separate IPPOL, MPPOL and normal oral keratinocytes non-invasively in vivo, we conducted an unbiased screen of their conditioned medium. MPPOL keratinocytes showed elevated levels of branch-chain amino acid, lipid, prostaglandin, and glutathione metabolites, some of which could potentially be converted into volatile compounds by oral bacteria and detected in breath analysis. Extracellular metabolites were generally depleted in IPPOL, and only six were elevated, but some metabolites distinguishing IPPOL from MPPOL have been associated with progression to oral squamous cell carcinoma (OSCC) in vivo. One of the metabolites elevated in IPPOL relative to the other groups, citrate, was confirmed by targeted metabolomics and, interestingly, has been implicated in cancer growth and metastasis. Although our investigation is preliminary, some of the metabolites described here are detectable in the saliva of oral cancer patients, albeit at a more advanced stage, and could eventually help detect oral cancer development earlier.

Characteristics of experimental resin-modified glass-ionomer cements, containing alternate monomers to HEMA.

OBJECTIVE: Resin-modified glass ionomer cements (RMGICs) present several advantages (e.g. fluoride release), but their reported cytotoxicity has been associated with hydroxyethyl methacrylate (HEMA) monomer release. Therefore, different monomers were tested for use in RMGICs in order to improve their biocompatibility and reduce monomer release. METHODS: Eight experimental liquid compositions were prepared replacing different percentages of HEMA (conventional monomer used in commercial RMGICs) with hydroxypropyl-methacrylate (HPM) and/or tetrahydrofurfuryl-methacrylate (THFM), which are known to have better biocompatibility. Moreover, two commercial materials (Fuji-Plus and RelyX) and two compositions, based on these (home), were included as controls. Monomer release of all materials (commercial, home and experimental) were tested using high-performance liquid chromatography (HPLC) methods after immersing discs in deionized-water (DW) or ethanol:DW. Cytotoxicity of the materials extracts was tested on normal human oral fibroblast line (NHOF-1) using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. RESULTS: Three experimental materials containing THFM (F3, R3 and R4) showed less or similar monomer release compared to corresponding commercial products. Furthermore, two experimental materials (F3 and F4) showed similar effects on NHOF-1 cells compared to the negative control medium. SIGNIFICANCE: The lower monomer release and higher cell viability of some experimental THFM compositions are encouraging. THFM partially replacing HEMA is potentially a suitable alternative for producing biocompatible RMGICs.

Oxidative Stress-Mediated YAP Dysregulation Contributes to the Pathogenesis of Pemphigus Vulgaris.

Pemphigus Vulgaris (PV) is a life-threatening autoimmune disease manifested with blisters in the skin and mucosa and caused by autoantibodies against adhesion protein desmoglein-3 (Dsg3) expressed in epithelial membrane linings of these tissues. Despite many studies, the pathogenesis of PV remains incompletely understood. Recently we have shown Dsg3 plays a role in regulating the yes-associated protein (YAP), a co-transcription factor and mechanical sensor, and constraining reactive oxygen species (ROS). This study investigated the effect of PV sera as well as the anti-Dsg3 antibody AK23 on these molecules. We detected elevated YAP steady-state protein levels in PV cells surrounding blisters and perilesional regions and in keratinocytes treated with PV sera and AK23 with concomitant transient ROS overproduction. Cells treated with hydrogen peroxide also exhibited augmented nuclear YAP accompanied by reduction of Dsg3 and α-catenin, a negative regulator of YAP. As expected, transfection of α-catenin-GFP plasmid rendered YAP export from the nucleus evoked by hydrogen peroxide. In addition, suppression of total YAP was observed in hydrogen peroxide treated cells exposed to antioxidants with enhanced cell-cell adhesion being confirmed by decreased fragmentation in the dispase assay compared to hydrogen peroxide treatment alone. On the other hand, the expression of exogenous YAP disrupted intercellular junction assembly. In contrast, YAP depletion resulted in an inverse effect with augmented expression of junction assembly proteins, including Dsg3 and α-catenin capable of abolishing the effect of AK23 on Dsg3 expression. Finally, inhibition of other kinase pathways, including p38MAPK, also demonstrated suppression of YAP induced by hydrogen peroxide. Furthermore, antioxidant treatment of keratinocytes suppressed PV sera-induced total YAP accumulation. In conclusion, this study suggests that oxidative stress coupled with YAP dysregulation attributes to PV blistering, implying antioxidants may be beneficial in the treatment of PV.

Prognostic and clinicopathological significance of podoplanin immunoexpression in oral and oropharyngeal squamous cell carcinoma: A systematic review.

BACKGROUND: Podoplanin (PDPN) is a glycoprotein associated with epithelial-mesenchymal transition, invasion, and metastasis in several types of malignancies, including oral and oropharyngeal squamous cell carcinoma (OSCC). The aim of this systematic review (SR) was to summarize and critically appraise the available evidence about the association between PDPN immunoexpression and clinicopathological features and its utility as a prognostic marker in OSCC. METHODS: Five electronic databases and three gray literature databases were searched for immunohistochemical studies, which were selected in a two-phase process. RESULTS: From 721 records identified, 22 cohort and seven analytical cross-sectional studies were included. Few studies reported that PDPN expression was associated with poorer survival rates in OSCC: overall survival = 4/12, disease-free survival = 4/7, and cancer-specific survival = 2/4 studies. Positive associations were most frequently reported on lymph node involvement, higher histopathological grade, and advanced clinical stages. CONCLUSION: Within limitations of this SR, PDPN may be associated with lymph node involvement, histopathological grade, and clinical stage of OSCC. Current evidence suggests that PDPN could be a useful prognostic marker for OSCC that needs further exploration.

Induction of p53 in keratinocyte cultures treated with Behçet's patient sera.

BACKGROUND: Behçet's disease (BD) is a rare, multisystem vasculitis disease characterized by recurrent orogenital ulcerations with its etiology remained unclear. The transcription factor p53 has been reported to be upregulated in some autoimmune diseases, such as lupus erythematosus, dermatomyositis, and psoriasis. However, little is known about its alteration in BD. METHODS: Keratinocyte cultures of both skin and oral origins were treated sera of 18 Behcet patients for 24 hours and analyzed by indirect immunofluorescence for p53 expression. The specificity of p53 expression was confirmed by siRNA-mediated p53 knockdown and the serum IgG removal studies. The expression of p53 levels was quantitatively analyzed with ImageJ. RESULTS: It was shown that the expression of p53 is increased in skin and oral keratinocyte cell lines, in both the nucleus and cytoplasm of cells treated with patient sera compared to controls. Either p53 knockdown or IgG removal results in a reduction of p53 levels relative to cells treated with patient sera without p53 knockdown or IgG depletion. CONCLUSIONS: This in vitro study provides the first evidence that BD sera can induce the p53 expression in keratinocytes that may have implications in Behcet pathogenesis.

Desmoglein-3 acts as a pro-survival protein by suppressing reactive oxygen species and doming whilst augmenting the tight junctions in MDCK cells.

Kidney disease prevalence increases with age, with a common feature of the disease being defects in the epithelial tight junctions. Emerging evidence suggests that the desmosomal adhesion protein Desmoglein-3 (Dsg3) functions beyond the desmosomal adhesion and plays a role in regulating the fundamental pathways that govern cell fate decisions in response to environmental chemical and mechanical stresses. In this study, we explored the role of Dsg3 on dome formation, reactive oxygen species (ROS) production and transepithelial electrical resistance (TER) in MDCK cells, a kidney epithelial cell model widely used to study cell differentiation and tight junction formation and integrity. We show that overexpression of Dsg3 constrained nuclear ROS production and cellular doming in confluent cell cultures and these features coincided with augmented TER and enhanced tight junction integrity. Conversely, cells expressing dominant-negative Dsg3ΔC mutants exhibited heightened ROS production and accelerated doming, accompanied by increased apoptosis, as well as cell proliferation, with massive disruption in F-actin organization and accumulation, and alterations in tight junctions. Inhibition of actin polymerization and protein synthesis was able to sufficiently block dome formation in mutant populations. Taken together, these findings underscore that Dsg3 has a role in controlling cellular viability and differentiation as well as the functional integrity of tight junctions in MDCK cells.

Analysis of head and neck carcinoma progression reveals novel and relevant stage-specific changes associated with immortalisation and malignancy.

We report changes in the genomic landscape in the development of head and neck squamous cell carcinomas HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Likely pathological mutations predominantly involved a relatively small set of genes reported previously (TP53, KMT2D, CDKN2A, PIK3CA, NOTCH1 and FAT1) but also other predicted cancer drivers (MGA, PABPC3, NR4A2, NCOR1 and MACF1). Notably, all these mutations arise early and are present in PPOLs. The most frequent genetic changes, which follow acquisition of immortality and loss of senescence, are of consistent somatic copy number alterations (SCNAs) involving chromosomal regions enriched for genes in known and previously unreported cancer-related pathways. We mapped the evolution of SCNAs in HNSCC progression. One of the earliest SCNAs involved deletions of CSMD1 (8p23.2). CSMD1 deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines. Modulation of CSMD1 in cell lines revealed significant suppression of proliferation and invasion by forced expression, and significant stimulation of invasion by knockdown of expression. Known cancer drivers NOTCH1, PPP6C, RAC1, EIF4G1, PIK3CA showed significant increase in frequency of SCNA in transition from PPOLs to HNSCC that correlated with their expression. In the later stages of progression, HNSCC with and without nodal metastases showed some clear differences including high copy number gains of CCND1, hsa-miR-548k and TP63 in the metastases group.

Senescence-Derived Extracellular Molecules as Modulators of Oral Cancer Development: A Mini-Review.

Oral cancers are predominantly oral squamous cell carcinomas (OSCCs) derived from keratinocytes, and there is now very detailed knowledge of the genetics and molecular biology of the epithelial tumourigenic component of these cancers, including the identification of cancer stem or tumour-initiating cells. Several key genetic alterations have been identified including the near ubiquitous loss of the CDKN2A/p16INK4A and p53 pathways and telomerase activation, together with frequent inactivation of the NOTCH1 canonical pathway either by somatic genetic alterations or by the presence of human papilloma virus. There is also evidence that OSCCs arise from a 'field' of altered cells and that malignant conversion takes place pre-dominantly at the microscopic level. However, in the last decade, it has been realised that tumour development and progression are influenced by the cells of the microenvironment with cross-talk between the epithelial (tumour) and mesenchymal components. OSCCs, especially those that have bypassed cellular senescence, produce an array of proteins and metabolites that induce cellular senescence in the normal surrounding cells; indeed, senescence is a common property of cancer-associated fibroblasts (CAFs). Cellular senescence is defined as an irreversible cell cycle arrest and is associated with the release of molecules known as the senescence-associated secretory phenotype that can selectively promote the growth of pre-neoplastic keratinocytes (osteopontin) and cancer invasion (transforming growth factor ß, matrix metalloproteinases, interleukin 6 and lactate). In addition, both old and new work has shown that keratinocytes harbouring NOTCH loss-of-function mutations that lead to defective keratinocyte differentiation and loss of squamous epithelial barrier function may act as a tumour-promoting stimulus for initiated cells harbouring RAS pathway mutations by activating a wound response in the tumour mesenchyme. Thus, not all keratinocytes in the tumour tissue may be tumourigenic and may instead act as promoters of tumour growth and progression analogous to the much-studied CAFs which co-evolve with the genetically altered tumourigenic cells. This new data is discussed in relation to attempts to develop novel non-invasive diagnostics and therapeutics for oral cancer.

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 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.

The secreted protein S100A7 (psoriasin) is induced by telomere dysfunction in human keratinocytes independently of a DNA damage response and cell cycle regulators.

BACKGROUND: Replicative senescence is preceded by loss of repeat sequences of DNA from the telomeres that eventually leads to telomere dysfunction, the accumulation of irreparable DNA double strand breaks and a DNA damage response (DDR). However, we have previously reported that whilst telomere dysfunction in human keratinocytes is associated with a permanent cell cycle arrest, the DDR was very weak and transcriptional profiling also revealed several molecules normally associated with keratinocytes terminal differentiation, including S100A7 (psoriasin). RESULTS: We show here that S100A7 and the closely related S100A15 (koebnerisin) are not induced by repairable or irreparable DSBs, ruling out the hypotheses that these genes are induced either by the low DDR observed or by non-specific cell cycle arrest. We next tested whether S100A7 was induced by the cell cycle effectors ARF (p14(ARF)), CDKN2A (p16(INK4A)) and TP53 (p53) and found that, although all induced a similar level of acute and permanent cell cycle arrest to telomere dysfunction, none induced S100A7 (except p53 over-expression at high levels), showing that cell cycle arrest is not sufficient for its induction. The closely related transcript S100A15 was also upregulated by telomere dysfunction, to a similar extent by p16(INK4A) and p53 and to a lesser extent by p14(ARF). CONCLUSIONS: Our results show that mere cell cycle arrest, the upregulation of senescence-associated cell cycle effectors and DNA damage are not sufficient for the induction of the S100 transcripts; they further suggest that whilst the induction of S100A15 expression is linked to both telomere-dependent and -independent senescence, S100A7 expression is specifically associated with telomere-dependent senescence in normal keratinocytes. As both S100A7 and S100A15 are secreted proteins, they may find utility in the early detection of human keratinocyte telomere dysfunction and senescence.

Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2.

The long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs)-eicosapentaenoic acid (EPA) and its metabolite docosahexaenoic acid (DHA)-inhibit cancer formation in vivo, but their mechanism of action is unclear. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation and inhibition have both been associated with the induction of tumour cell apoptosis by n-3 PUFAs. We show here that low doses of EPA, in particular, inhibited the growth of premalignant and malignant keratinocytes more than the growth of normal counterparts by a combination of cell cycle arrest and apoptosis. The growth inhibition of the oral squamous cell carcinoma (SCC) lines, but not normal keratinocytes, by both n-3 PUFAs was associated with epidermal growth factor receptor (EGFR) autophosphorylation, a sustained phosphorylation of ERK1/2 and its downstream target p90RSK but not with phosphorylation of the PI3 kinase target Akt. Inhibition of EGFR with either the EGFR kinase inhibitor AG1478 or an EGFR-blocking antibody inhibited ERK1/2 phosphorylation, and the blocking antibody partially antagonized growth inhibition by EPA but not by DHA. DHA generated more reactive oxygen species and activated more c-jun N-terminal kinase than EPA, potentially explaining its increased toxicity to normal keratinocytes. Our results show that, in part, EPA specifically inhibits SCC growth and development by creating a sustained signalling imbalance to amplify the EGFR/ERK/p90RSK pathway in neoplastic keratinocytes to a supraoptimal level, supporting the chemopreventive potential of EPA, whose toxicity to normal cells might be reduced further by blocking its metabolism to DHA. Furthermore, ERK1/2 phosphorylation may have potential as a biomarker of n-3 PUFA function in vivo.

The induction of apoptosis in pre-malignant keratinocytes by omega-3 polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is inhibited by albumin.

The long chain omega-3 polyunsaturated fatty acids (PUFA) have been reported to exert anti-cancer effects. At this study we tested the effect of the omega-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on pre-malignant keratinocytes growth in the well-characterised human pre-malignant epidermal cell line, HaCaT and attempted to identify a PUFA serum antagonist. Both EPA and DHA inhibited HaCaT growth and induced apoptosis. At the 10% (v/v) foetal bovine serum (FBS) medium, limited growth inhibition (3-20% for 50µM DHA and EPA respectively) and negligible apoptosis were observed with PUFA use. However, at 3% (v/v) FBS medium, 30-50µM of PUFA caused impressive levels of growth inhibition (82-83% for 50µM DHA and EPA respectively) and increase of apoptosis (8-19% increase in 72h). None of the numerous serum growth factors present in FBS or the antioxidant n-tert-butyl-α-phenylnitrone could inhibit the PUFA-induced cytotoxicity. In contrast, bovine and human albumin (0.1-0.3%, w/v) significantly antagonized the growth inhibitory and apoptosis-inducing effects of PUFA. In conclusion, we have shown for the first time that omega-3 PUFA inhibit the growth and induce apoptosis of pre-malignant keratinocytes and identified albumin as a major antagonistic factor in serum that could limit their effectiveness at pharmacologically-achievable doses.

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.