Elisabetta Falcieri

Ultraviolet B (UVB) Irradiation-Induced Apoptosis in Various Cell Lineages in Vitro

Ultraviolet B (UVB) radiation acts as a strong apoptotic trigger in many cell types, in tumor and normal cells. Several studies have demonstrated that UVB-induced cell death occurs through the generation of reactive oxygen species. The consequent oxidative stress includes the impairment of cellular antioxidants, the induction of DNA damage and the occurrence of apoptosis. In this review, we investigated UVB apoptotic action in various cell models by using ultrastructural, molecular and cytofluorimetric techniques. Myeloid leukemia HL-60, T-lymphoblastoid Molt-4 and myelomonocytic U937 human cells, generally affected by apoptotic stimuli, were studied. Human chondrocytes and C2C12 skeletal muscle cells, known to be more resistant to damage, were also considered. All of them, when exposed to UVB radiation, revealed a number of characteristic apoptotic markers. Membrane blebbing, cytoplasm shrinkage and chromatin condensation were detected by means of electron microscopy. DNA cleavage, investigated by using agarose gel electrophoresis and TUNEL reaction, was observed in suspended cells. Differently, in chondrocytes and in skeletal muscle cells, oligonucleosomic DNA fragmentation did not appear, even if a certain TUNEL positivity was detected. These findings demonstrate that UVB radiation appears to be an ideal tool to study the apoptotic behavior.

Tyrosol prevents apoptosis in irradiated keratinocytes

BACKGROUND Phenolic compounds, the biggest group of natural antioxidants, have attracted much attention due to their known and wide-ranging biological activities, as well as to their health effects. In particular, regardless their antioxidant activity, they play a key role in the control of several inflammation-associated processes as well as in improving antioxidant defense system. In an our previous work we have demonstrated the ability of Hydroxytyrosol, an ortho-diphenolic compound, essential component of oleuropein, in preventing apoptotic cell death induced by UVB radiation in HaCaT cell lines in vitro. In olive oil, besides Hydroxytyrosol, there are appreciable amounts of Tyrosol and its secoiridoid derivatives. OBJECTIVE It has been well established that Tyrosol has a significantly lower antioxidant activity than Hydroxytyrosol, but despite this, recent studies suggest that Tyrosol exerts a powerful protective effect against oxidative injuries in cell systems and that it is able to improve the intracellular antioxidant defenses. MATERIALS AND METHODS Here, Tyrosol effect has been evaluated in HaCaT cells exposed to UVB radiation by means of morphological and molecular analyses. RESULTS Our study revealed the polyphenol ability in reducing apoptotic markers and in protecting HaCaT cells from damage. CONCLUSION These findings suggest an important role of Tyrosol in protecting cells from apoptotic cell death and encourage the use of this phytochemical as biological ingredient in topical preparations as possible tool to prevent skin damage.

α-Actinin involvement in Z-disk assembly during skeletal muscle C2C12 cells in vitro differentiation.

α-Actinin is involved in the assembly and maintenance of muscle fibers. α-Actinin is required to cross-link actin filaments and to connect the actin cytoskeleton to the cell membrane and it is necessary for the attachment of actin filaments to Z-disks in skeletal muscle fibers and to dense bodies in smooth muscle ones. In addition to its mechanical role, sarcomeric α-actinin interacts with proteins involved in a variety of signaling and metabolic pathways. The aim of this work is to monitor Z-disk formation, in order to clear up the role of sarcomeric α-actinin in undifferentiated stage, after 4 days of differentiation (intermediate differentiation stage) and after 7 days of differentiation (fully differentiated stage). For this purpose, C2C12 murine skeletal muscle cells, grown in vitro, were analyzed at three time points of differentiation. Confocal laser scanner microscopy and transmission electron microscopy have been utilized for α-actinin immunolocalization. Both techniques reveal that in undifferentiated cells labeling appears uniformly distributed in the cytoplasm with punctate α-actinin Z-bodies. Moreover, we found that when differentiation is induced, α-actinin links at first membrane-associated proteins, then it aligns longitudinally across the cytoplasm and finally binds actin, giving rise to Z-disks. These findings evidence α-actinin involvement in sarcomeric development, suggesting for this protein an important role in stabilizing the muscle contractile apparatus.

Three-dimensional apoptotic nuclear behavior analyzed by means of Field Emission in Lens Scanning Electron Microscope.

Apoptosis is an essential biological function required during embryogenesis, tissue home-ostasis, organ development and immune system regulation. It is an active cell death pathway involved in a variety of pathological conditions. During this process cytoskeletal proteins appear damaged and undergo an enzymatic disassembling, leading to formation of apoptotic features. This study was designed to examine the three-dimensional chromatin behavior and cytoskeleton involvement, in particular actin re-modeling. HL-60 cells, exposed to hyperthermia, a known apoptotic trigger, were examined by means of a Field Emission in Lens Scanning Electron Microscope (FEISEM). Ultrastructural observations revealed in treated cells the presence of apoptotic patterns after hyperthermia trigger. In particular, three-dimensional apoptotic chromatin rearrangements appeared involving the translocation of filamentous actin from cytoplasm to the nucleus. FEISEM immunogold techniques showed actin labeling and its precise three-dimensional localization in the diffuse chromatin, well separated from the condensed one. The actin presence in dispersed chromatin inside the apoptotic nucleus can be considered an important feature, indispensable to permit the apoptotic machinery evolution.

Protective effect of different antioxidant agents in UVB-irradiated keratinocytes

Skin cells can respond to UVB-induced damage either by tolerating it, or restoring it through antioxidant activation and DNA repair mechanisms or, ultimately, undergoing programmed cell death, when damage is massive. Nutritional factors, in particular, food antioxidants, have attracted much interest because of their potential use in new preventive, protective, and therapeutic strategies for chronic degenerative diseases, including skin inflammation and cancer. Some polyphenols, present in virgin olive oil, well tolerated by organism after oral administration, show a variety of pharmacological and clinical benefits such as antioxidant, anti-cancer, anti-inflammatory, and neuro-protective activities. Here, the protective effects of antioxidant compounds against UV-induced apoptosis have been described in HaCaT cell line. Human keratinocytes were pre-treated with antioxidants before UVB exposure and their effects have been evaluated by means of ultrastructural analyses. After UVB radiation, a known cell death trigger, typical apoptotic features, absent in control condition and in antioxidant alone-treated cells, appear. An evident numerical decrease of ultrastructural apoptotic patterns and TUNEL positive nuclei can be observed when natural antioxidants were supplied before cell death induction. These data have been confirmed by molecular investigation of caspase activity. In conclusion, this paper highlights antioxidant compound ability to prevent apoptotic cell death in human keratinocytes exposed to UVB, suggesting, for these molecules, a potential role in preventing skin damage.

Prevention of UVB radiation-induced cell death: “in vitro” studies

The ultraviolet component of sun light consists of UVA, UVB and UVC rays. UVB radiation represents an environmental hazard because of its role in skin aging, cancer and infection exacerbation. UVB stimulate the production of reactive oxygen species (ROS) in epidermal cells, resulting in skin lesions, accelerating aging and eliciting malignancies. At least 50% of UVB-induced damage is attributable to the formation of reactive ROS which cause cellular lesions if antioxidant defence mechanisms are down-regulated. Thus, exogenous supplementation of antioxidants may be an effective strategy to reduce or prevent skin damage. In the last years, we demonstrated the antioxidant effects of melatonin (Mel) (Luchetti et al., 2006) and, more recently of hydroxytyrosol (HyT) and its derivatives (Burattini et al., 2013) in hemopoietic human cells exposed to pro-oxidants. Therefore, in this project we propose to evaluate the antioxidant and/or anti-apoptotic effect of Mel and HyT in HaCaT human keratinocytes exposed to UVB. Keratinocytes in the non-irradiated condition are morphologically similar in Mel- and HyT-treated and untreated group. TUNEL reaction appears negative in both conditions, as well as in control. UVB radiation induces a significant decrease in cell confluence, with a diffuse cell detachment and the appearance of rounding and blebbed cells. TUNEL reaction evidences several nuclei with DNA fragmentation in UVB treated keratinocytes. In addition, cell viability evaluated by means of supravital propidium iodide (PI) evidences a diffuse staining positivity. Pre-treatment with Mel or HyT before UVB exposure is able to reduce cell death. In conclusion, HyT and Mel evidence an intringuing capability to prevent cell death in keratinocytes too. They could so represent a potential tool in skin protection from UVB radiation.

α-Actinin behavior during C2C12 along differentiation

α-Actinin is a cytoskeletal actin-binding protein (Ogura et al., 2009) that provides structural integrity of the sarcomeres and is located in the skeletal muscle Z-lines. It creates cross-links between actin filaments and, besides, it contributes to cytoskeleton organization and muscle contraction (Sjoblom et al., 2008). The aim of this work was to clear up the behavior of sarcomeric α-actinin in Z-lines formation during myogenic differentiation. For this purpose, C2C12 cells were analyzed at 0, 3, 7 days of differentiation, monitoring cell maturation and viability by means of inverted microscopy. Immuno-labeling of sarcomeric α-actinin was investigated both at CLSM and at TEM, using a mouse anti-α-actinin antibody followed by a FITC-conjugated goat antimouse or a 10nm colloidal gold conjugated anti-mouse antibody (Ferri et al., 2009), respectively. Immunofluorescence analysis reveals that, when differentiation is induced, initially α-actinin colocalizes with membrane-associated proteins, then it aligns longitudinally across the cytoplasm and, finally, it binds actin, giving rise to Z-lines. Immunogold study generally evidences a cytoplasmic and nuclear positivity, indicating a role for α-actinin in signaling, chromatin remodeling and in shuttle between these compartments (Dingova et al., 2009; Lin et al., 2010). This study shows an α-actinin specific distribution and dynamic organization along the differentiation process.

Skeletal muscle cell death induced by physical agents

Apoptosis plays a pivotal role in the deletion of unwanted, damaged, or infected cells in multicellular organisms, as well as in development and tissue homeostasis, cell differentiation, and proliferation. In skeletal muscle cells it is unique for several reasons. First, skeletal muscle fibre is multinucleated. So muscle cell death is correlated to a loss of gene expression within the local myonuclear domain, potentially leading to muscle atrophy. In addition, skeletal muscle is a plastic tissue capable of changing its mitochondrial content and/or composition in response to chronic alterations in muscle use or disuse (Siu et al., 2009). Most of the research evidenced that many of the external apoptotic stimuli activate signaling pathways that converge on the mitochondria, determining cell death (Adhietty et al., 2008). Physical triggers such as UVB (D’Emilio et al., 2010), hyperthermia (Lee et al., 2011) and hypothermia (Pizanis et al., 2011) induced cell death by mitochondrial pathways in various cell types. In addition also low pH usually induced DNA damage in other cell lines (Xiao et al., 2003). The aim of this work is to investigate in vitro skeletal muscle cell death appearing after exposure to physical triggers, by means of TUNEL reaction, analysed at confocal microscope, and of electron microscopy. C2C12 myoblasts and myotubes, grown as previously reported (D’Emilio et al., 2010), were exposed to UV-B (312nm) for 30 min, hyperthermia 45°C for 1h and hypothermia (2-6°C) and low pH (5) for 4h. All treatments were followed by 2h recovery. Control cell evidentiated a good morphology and appeared negative to TUNEL reaction. UVB - treated sample presented nuclear features suggest apoptosis both at electron and confocal microscopy and in undifferentiated and differentiated conditions. Hyperthermia induced both apoptosis and necrosis with cell rounding and a certain positivity to TUNEL reaction both in myoblasts and myotubes. After hypothermia apoptosis was observed in some cells, but the majority appeared similar to the control, so evidentiating a scarce response. Cells treated with low had swollen nuclei, sometimes showing a thin film of condensed chromatin, and occasionally TUNEL-positive. In all conditions cytoplasm vacuolisaton and autophagic vacuole increase appeared. These findings suggest that skeletal muscle cells seem to be sensitive to physical agents induced cell death.