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Skeletal muscle cell death induced by physical agents.
Full text linkApoptosis 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
Cytoprotective effects of melatonin in C2C12 skeletal muscle cells: a multiple technical approach.
Full text linkMelatonin has a wide range of physiological functions including protection against oxidative stress, which is carried out through its ability to act as a free radical scavenger and to stimulate antioxidant enzyme production (Allegra et al., 2003). Oxidative stress is a major player in initiating apoptosis in skeletal muscle, as well as in other tissues. Apoptosis is essential for skeletal muscle development and homeostasis; nevertheless, its misregulation has been frequently observed in various myopathies (Loro et al., 2010). Several authors demonstrated that melatonin exerts antiapoptotic actions in various cell models (Hibaoui et al., 2009) and our previous studies evidenced that it prevents apoptosis induced by UV-B and H2O2 in U937 cells (Luchetti et al., 2006; Salucci et al., 2010). In this work, melatonin activity has been investigated in C2C12 cells, after apoptotic chemical treatments. Myoblasts and myotubes were pre-treated with melatonin and then exposed to H2O2, cisplatin, etoposide and staurosporine. Data, obtained by means of TEM and TUNEL-CLSM, show that melatonin prevents apoptosis induced by H2O2, cisplatin and etoposide. Differently, staurosporine-induced apoptosis is not inhibited, probably because this trigger has a mechanism of action different from free radical increase. These results confirm melatonin ability to act as an antioxidant and anti-apoptotic molecule, thus suggesting a possible therapeutic strategy for myophaties involving apoptosis misregulation
Prevention of UVB radiation-induced cell death: “in vitro” studies. August 25-30, 2013, Proceedings pag. 95-96.
Full text linkThe 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
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