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Radiotherapy is widely used for the treatment of breast cancer. However, we have shown that ionizing radiation can provoke premature senescence in breast stromal cells. In particular, breast stromal fibroblasts can become senescent after irradiation both in vitro and in vivo and they express an inflammatory phenotype and an altered profile of extracellular matrix components, thus facilitating tumor progression. Adipose-derived stem cells (ASCs) represent another major component of the breast tissue stroma. They are multipotent cells and due to their ability to differentiate in multiple cell lineages they play an important role in tissue maintenance and repair in normal and pathologic conditions. Here, we investigated the characteristics of human breast ASCs that became senescent prematurely after their exposure to ionizing radiation. We found decreased expression levels of the specific mesenchymal cell surface markers CD105, CD73, CD44, and CD90. In parallel, we demonstrated a significantly reduced expression of transcription factors regulating osteogenic (i.e., RUNX2), adipogenic (i.e., PPARγ), and chondrogenic (i.e., SOX9) differentiation; this was followed by an analogous reduction in their differentiation capacity. Furthermore, they overexpress inflammatory markers, that is, IL-6, IL-8, and ICAM-1, and a catabolic phenotype, marked by the reduction of collagen type I and the increase of MMP-1 and MMP-13 expression. Finally, we detected changes in proteoglycan expression, for example, the upregulation of syndecan 1 and syndecan 4 and the downregulation of decorin. Notably, all these alterations, when observed in the breast stroma, represent poor prognostic factors for tumor development. In conclusion, we showed that ionizing radiation-mediated prematurely senescent human breast ASCs have a decreased differentiation potential and express specific changes adding to the formation of a permissive environment for tumor growth.

SummaryBackgroundCyclic tensile stretching (CTS) induces osteoblastic differentiation of periodontal ligament fibroblasts (PDLF). On the other hand, increased concentrations of tumour necrosis factor-α (TNF-α) are found in inflammatory conditions, leading to periodontal disease and tooth loss. Accordingly, our aim was to investigate the short- and long-term effect of TNF-α on the response of human PDLF to CTS and its implication on osteoblastic differentiation.MethodsPDLF were either pre-incubated for 4 hours or were repeatedly exposed to TNF-α for up to 50 days and then subjected to CTS. Gene expression was determined by quantitative real-time polymerase chain reaction. Activation of mitogen-activated protein kinase (MAPK) was monitored by western analysis and cell proliferation by bromodeoxyuridine incorporation. Intracellular reactive oxygen species were determined by the 2´, 7´-dichlorofluorescein-diacetate assay and osteoblastic differentiation by Alizarin Red-S staining after an osteo-inductive period of 21 days.ResultsCTS of PDLF induced an immediate upregulation of the c-fos transcription factor and, further downstream the overexpression of alkaline phosphatase and osteopontin, two major osteoblast marker genes. A 4-hour pre-incubation with TNF-α repressed these effects. Similarly, long-term propagation of PDLF along with TNF-α diminished their osteoblastic differentiation capacity and suppressed cells’ CTS-elicited responses. The observed phenomena were not linked with TNF-α-induced premature senescence or oxidative stress. While CTS induced the activation of MAPKs, involved in mechanotransduction, TNF-α treatment provoked a small delay in the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase.ConclusionIncreased concentrations of TNF-α, such as those recorded in many inflammatory diseases, suppress PDLF’s immediate responses to mechanical forces compromising their osteoblastic differentiation potential, possibly leading to tissue’s impaired homeostasis.

Simple Summary Ionizing radiation (a typical remedy for breast cancer) results in the premature senescence of the adjacent to the neoplastic cells stromal fibroblasts. Here, we showed that these senescent fibroblasts are characterized by the down-regulation of the small leucine-rich proteoglycan decorin, a poor prognostic factor for the progression of the disease. Decorin down-regulation is mediated by secreted growth factors in an autocrine and paracrine (due to the interaction with breast cancer cells) manner, with bFGF and VEGF being the key players of this regulation in young and senescent breast stromal fibroblasts. Autophagy activation increases decorin mRNA levels, indicating that impaired autophagy is implicated in the reduction in decorin in this cell model. Decorin down-regulation acts additively to the already tumor-promoting phenotype of ionizing radiation-induced prematurely senescent human stromal fibroblasts, confirming that stromal senescence is a side-effect of radiotherapy that should be taken into account in the design of anticancer treatments. Abstract Down-regulation of the small leucine-rich proteoglycan decorin in the stroma is considered a poor prognostic factor for breast cancer progression. Ionizing radiation, an established treatment for breast cancer, provokes the premature senescence of the adjacent to the tumor stromal fibroblasts. Here, we showed that senescent human breast stromal fibroblasts are characterized by the down-regulation of decorin at the mRNA and protein level, as well as by its decreased deposition in the pericellular extracellular matrix in vitro. Senescence-associated decorin down-regulation is a long-lasting process rather than an immediate response to γ-irradiation. Growth factors were demonstrated to participate in an autocrine manner in decorin down-regulation, with bFGF and VEGF being the critical mediators of the phenomenon. Autophagy inhibition by chloroquine reduced decorin mRNA levels, while autophagy activation using the mTOR inhibitor rapamycin enhanced decorin transcription. Interestingly, the secretome from a series of both untreated and irradiated human breast cancer cell lines with different molecular profiles inhibited decorin expression in young and senescent stromal fibroblasts, which was annulled by SU5402, a bFGF and VEGF inhibitor. The novel phenotypic trait of senescent human breast stromal fibroblasts revealed here is added to their already described cancer-promoting role via the formation of a tumor-permissive environment.

Senescent fibroblasts are characterized by their inability to proliferate and by a pro-inflammatory and catabolic secretory phenotype, which contributes to age-related pathologies. Furthermore, senescent fibroblasts when cultured under classical conditions in vitro are also characterized by striking morphological changes, i.e. they lose the youthful spindle-like appearance and become enlarged and flattened, while their nuclei from elliptical become oversized and highly lobulated. Knowing the strong relation between cell shape and function, we cultured human senescent fibroblasts on photolithographed Si/poly(vinyl alcohol) (PVA) micro-patterned surfaces in order to restore the classical spindle-like geometry and subsequently to investigate whether the changes in senescent cells' morphology are the cause of their functional alterations. Interestingly, under these conditions senescent cells' nuclei do not revert to the classical elliptical phenotype. Furthermore, enforced spindle-shaped senescent cells retained their deteriorated proliferative ability, and maintained the increased gene expression of the cell cycle inhibitors p16Ink4a and p21Waf1. In addition, Si/PVA-patterned-grown senescent fibroblasts preserved their senescence-associated phenotype, as evidenced by the overexpression of inflammatory and catabolic genes such as IL6, IL8, ICAM1 and MMP1 and MMP9 respectively, which was further manifested by an intense downregulation of fibroblasts' most abundant extracellular matrix component Col1A, compared to their young counterparts. These data indicate that the restoration of the spindle-like shape in senescent human fibroblasts is not able to directly alter major functional traits and restore the youthful phenotype.

The feruloyl esterases FaeA1, FaeA2, FaeB1, FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464 were used as biocatalysts for the transesterification of vinyl ferulate (VFA) with Larabinose in detergentless microemulsions. The effect of parameters such as the microemulsion composition, the substrate concentration, the enzyme load, the pH, the temperature and the agitation was investigated. FaeA1 offered the highest transesterification yield (52.2 ± 4.3%) after 8 h of incubation at 50 °C using 80 mM VFA, 55 mM L-arabinose and 0.02 mg FAE mL−1 in a mixture comprising of 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. The ability of L-arabinose ferulate (AFA) to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was significant (IC50 386.5 μM). AFA was not cytotoxic even at high concentrations (1 mM) however was found to be pro-oxidant at concentrations higher than 20 μM when the antioxidant activity was determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay in human skin fibroblasts.

After publication of the original article, authors found that there has been a minor mistake in the units of kcat and kcat/Km in Table 2. The units should be 103 min-1 g-1 FAE for kcat and mM-1 min-1 g-1 FAE for kcat/Km. This correction does not affect any conclusions drawn within the article.

Summary Objectives The aim of the present study was to investigate the impact of high glucose concentration on the response of human periodontal ligament fibroblasts (PDLFs) to cyclic tensile strain. Materials and Methods Human PDLFs were incubated under normal or high glucose conditions, and then were subjected to cyclic tensile stretching (8 per cent extension, 1 Hz). Gene expression was determined by quantitative real-time polymerase chain reaction. Intracellular reactive oxygen species (ROS) were determined by the 2’,7’-dichlorofluorescein-diacetate assay, activation of mitogen-activated protein kinase (MAPK) was monitored by western analysis and osteoblastic differentiation was estimated with Alizarin Red-S staining. Results Cyclic tensile stretching of PDLF leads to an immediate activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as to the increased expression of the transcription factor c-fos, known to regulate many osteogenesis-related genes. At later time points, the alkaline phosphatase and osteopontin genes were also upregulated. Hyperglycaemic conditions inhibited these effects. High glucose conditions were unable to increase ROS levels, but they increased the medium’s osmolality. Finally, increase of osmolality mimics the inhibitory effect of hyperglycaemia on MAPK activation, c-fos and osteoblast-specific gene markers’ upregulation, as well as osteogenic differentiation capacity. Conclusion Our findings indicate that under high glucose conditions, human PDLFs fail to adequately respond to mechanical deformation, while their strain-elicited osteoblast differentiation ability is deteriorated. The aforementioned effects are most probably mediated by the increased osmolality under hyperglycaemic conditions.

The genus Strychnos (Loganiaceae) is well-known as a rich source of various bioactive metabolites. In continuation of our phytochemical studies on plants from Amazonia, we examined Strychnosaff. darienensis, collected in Peru. This species has been traditionally used in South America and is still presently used as a drug by the Yanesha tribe in Peru. Phytochemical investigation of this plant led to the isolation and structure elucidation by &Nu, uclear&Mu, agnetic Resonance and High Resolution Mass Spectroscopy of 14 compounds that belong to the categories of phenolic acids [p-hydroxybenzoic acid (1) and vanillic acid (2)], flavonoids [luteolin, (3),3-O-methyl quercetin (4), strychnobiflavone (5), minaxin (6) and 3&rsquo, 4&rsquo, 7-trihydroxy-flavone (7)], lignans [syringaresinol-&beta, D-glucoside (8), balanophonin (9) and ficusal (10)] and alkaloids [venoterpine (11), 11-methoxyhenningsamine (12), diaboline (13) and 11-methoxy diaboline (14)]. The isolated flavonoids&mdash, a class known for its anti-aging activities&mdash, were further evaluated for their biological activities on normal human skin fibroblasts. Among them, only (6), and to a lesser extent (7), exhibited cytotoxicity at 100 &micro, g/ml. All five flavonoids suppressed intracellularreactive oxygen species (ROS) levels, either basal or following stimulation with hydrogen peroxide or both. Moreover, luteolin and strychnobiflavone protected skin fibroblasts against ultraviolet (UV)-irradiation-induced cell death. The isolated flavonoids could prove useful bioactive ingredients in the cosmetic industry.

Normal cells after a defined number of successive divisions or after exposure to genotoxic stresses are becoming senescent, characterized by a permanent growth arrest. In addition, they secrete increased levels of pro-inflammatory and catabolic mediators, collectively termed "senescence-associated secretory phenotype". Furthermore, senescent cells exhibit an altered expression and organization of many extracellular matrix components, leading to specific remodeling of their microenvironment. In this review we present the current knowledge on extracellular matrix alterations associated with cellular senescence and critically discuss certain characteristic examples, highlighting the ambiguous role of senescent cells in the homeostasis of various tissues under both normal and pathologic conditions.

Mechanical loading is considered to be a major parameter of the periodontal ligament (PDL) remodeling and differentiation. However, the molecular mechanisms that translate these forces to cellular responses are not fully elucidated. Especially, although aging affects PDL homeostasis, the role of cellular senescence on the activation of signaling pathways in periodontal ligament fibroblasts (PDLF) in response to mechanical stimulation has not been studied yet. Here, we present evidence showing that cyclic mechanical stimulation activates ERK, JNK and p38 MAPK in young (early-passage) human PDLF, in a RhoK-dependent manner. This response was found to be independent of the substratum (i.e. fibronectin or collagen) on which these cells grow. Stretching up-regulates also c-fos, a classical cellular response to mechanical deformation. Inhibition of ERK and JNK reduces, while that of p38 enhances stress-mediated c-fos expression. In addition, cyclic stretching stimulates the expression and activity of alkaline phosphatase (ALP), an early marker of osteoblastic differentiation. We have recently shown that senescent human PDLF have a significantly decreased expression of ALP, linked to an inability towards osteoblastic differentiation. Here, we found that senescent PDLF are able to respond to cyclic mechanical stretching by activating ERK, JNK and p38 MAPK, with similar kinetics compared to young cells, and by up-regulating c-fos and ALP expression and activity. However, even after stimulation, ALP levels in senescent cells are still much lower compared to the basal levels of their young counterparts, suggesting that senescence impairs the differentiation of human PDLF when subjected to cyclic mechanical deformation.

Objective: To compare the mechanotransduction caused by cyclic and static mechanical strains in human periodontal ligament fibroblasts (hPDLFs) cultured under identical conditions. Materials and methods: hPDLFs, originating from the same donors, were exposed either to cyclic or to static tensile strain using specially designed devices and under identical culture conditions. Activation of all members of mitogen-activated protein kinases (MAPKs) was monitored by western immunoblot analysis. Expression levels of immediate/early genes c-fos and c-jun were assessed with quantitative real-time polymerase chain reaction. Results: Time course experiments revealed that both types of stresses activate the three members of MAPK, that is ERK, p38, and JNK, with cyclic stress exhibiting a slightly more extended activation. Further downstream, both stresses upregulate the immediate/early genes c-fos and c-jun , encoding components of the activator protein-1 (AP-1), a key transcription factor in osteoblastic differentiation; again cyclic strain provokes a more intense upregulation. Six hours after the application of both strains, MAPK activation and gene expression return to basal levels. Finally, cells exposed to cyclic stress for longer periods are distributed approximately perpendicular to the axis of the applied strain, whereas cells exposed to static loading remain in a random orientation in culture. Conclusion: The findings of the present study indicate similar, although not identical, immediate/early responses of hPDLs to cyclic and static stretching, with cyclic strain provoking a more intense adaptive response of these cells to mechanical deformation.

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8–100 μM (IC50 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

Loss of teeth increases with age or after genotoxic treatments, like head and neck radiotherapy, due to periodontium breakdown. Periodontal ligament fibroblasts represent the main cell type in this tissue and are crucial for the maintenance of homeodynamics and for its regeneration. Here, we have studied the characteristics of human periodontal ligament fibroblasts (hPDLF) that became senescent after replicative exhaustion or after exposure to ionizing radiation, as well as their ability for osteoblastic differentiation. We found that senescent hPDLF express classical markers of senescence, as well as a catabolic phenotype, as shown by the decrease in collagen type I and the increase of MMP-2 expression. In addition, we observed a considerably decreased expression of the major transcription factor for osteoblastic differentiation, i.e. Runx2, a down-regulation which was found to be p53-dependent. In accordance to the above, senescent cells have a significantly decreased alkaline phosphatase gene expression and activity, as well as a reduced ability for osteoblastic differentiation, as found by Alizarin Red staining. Interestingly, cells from both type of senescence express similar characteristics, implying analogous functions in vivo. In conclusion, senescent hPDLF express a catabolic phenotype and express a significantly decreased ability towards an osteoblastic differentiation, thus probably affecting tissue development and integrity.

Biocompatibility testing of novel biomaterials employs a wide spectrum of various in vivo and in vitro tests. As in vitro testing can respond to the urgent need for screening the huge amount of new materials produced nowadays, the present chapter is focusing on the cell-based assay systems used for such tests and introduces briefly the most common in vitro methods for the evaluation of the cell response to biomaterials, with an emphasis on testing of cell death and cell proliferation. Advantages and disadvantages of these methods, some hints for the setup of the tests, as well as, novel trends in assay methodologies are also here discussed.

Cellular senescence, being the result of serial subculturing or of exogenous stresses, is considered to be a potent anticancer mechanism. However, it has been proposed that senescent cells may enhance the growth of adjacent malignant epithelial cells. On the other hand, exposure of tumors to repeated low doses of γ-irradiation is a common treatment regime. Nevertheless, γ-irradiation also affects the neighboring stromal cells and the interaction of the latter with cancer cells. Accordingly, in this study, we have exposed confluent cultures of human lung fibroblasts to repeated subcytotoxic doses of 4 Gy of γ-irradiation. We found that a single dose immediately activates a DNA damage response, leading to an intense, but reversible, cell cycle arrest. After a series of doses (total dose approximately 50 Gy) cellular senescence was accelerated, as shown by permanent growth arrest and the upregulation of specific biochemical and morphological senescence-associated markers. This process was found to be p53-dependent. Next, we studied the effect of these prematurely senescent cells on the growth of human malignant lung cell lines (A549 and H1299) and found that the presence of irradiation-mediated senescent cells strongly enhances the growth of these cancer cells in vitro and in immunocompromised (SCID) mice in vivo. This effect seems not to be related to an induction of epithelial-to-mesenchymal transdifferentiation but, to a significant extent, to the increased expression of matrix metalloproteases (MMPs), as a specific MMP inhibitor significantly restrains the growth of cancer in the presence of senescent fibroblasts. These findings indicate that lung fibroblasts that become senescent after ionizing radiation may contribute to lung cancer progression.