Valentina Baldassarri

Anti-apoptotic activity of hydroxytyrosol and hydroxytyrosyl laurate.

Hydroxytyrosol (HyT) is a polyphenol primarily released in olive mill wastewater and in olive oil. In animal and cell model studies, HyT and its metabolites have strong antioxidant and antimicrobial activities, as well as beneficial effects on the cardiovascular system and in several human diseases. Differently, many researchers reported that HyT down-regulates tumor cell viability and cell cycle progression, and induces reactive oxygen species (ROS) production and apoptosis. In this study we have investigated the effects of HyT and the corresponding ester hydroxytyrosyl laurate in U937 cells, a human monocytoid cell line, and in C2C12 myoblasts, a murine proliferating muscle cell model, after apoptotic death induction. Inverted, light and transmission electron microscopy have been utilized to characterize cell death patterns. H2O2, at the concentrations known to induce apoptosis, was utilized as cell death trigger. The results obtained show that laur-HyT has a protective antioxidant effect against H2O2 treatment, greater than HyT, so having a role in the prevention of apoptotic death in normal and tumor cells. These data suggest these compounds as good candidate for novel therapeutic strategies.

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.

Melatonin Prevents Chemical-Induced Haemopoietic Cell Death

Melatonin (MEL), a methoxyindole synthesized by the pineal gland, is a powerful antioxidant in tissues as well as within cells, with a fundamental role in ameliorating homeostasis in a number of specific pathologies. It acts both as a direct radical scavenger and by stimulating production/activity of intracellular antioxidant enzymes. In this work, some chemical triggers, with different mechanisms of action, have been chosen to induce cell death in U937 hematopoietic cell line. Cells were pre-treated with 100 μM MEL and then exposed to hydrogen peroxide or staurosporine. Morphological analyses, TUNEL reaction and Orange/PI double staining have been used to recognize ultrastructural apoptotic patterns and to evaluate DNA behavior. Chemical damage and potential MEL anti-apoptotic effects were quantified by means of Tali® Image-Based Cytometer, able to monitor cell viability and apoptotic events. After trigger exposure, chromatin condensation, micronuclei formation and DNA fragmentation have been observed, all suggesting apoptotic cell death. These events underwent a statistically significant decrease in samples pre-treated with MEL. After caspase inhibition and subsequent assessment of cell viability, we demonstrated that apoptosis occurs, at least in part, through the mitochondrial pathway and that MEL interacts at this level to rescue U937 cells from death.

Melatonin behavior in restoring chemical damaged C2C12 myoblasts.

It is known that, besides a wide range of functions, melatonin provides protection against oxidative stress, thanks to its ability to act, directly, as a free radical scavenger and, indirectly, by stimulating antioxidant enzymes production and mitochondrial electron transport chain efficiency. Oxidative stress is one of the major players in initiating apoptotic cell death 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 several myopathies, in sarcopenia, as well as in denervation and disuse. Melatonin activity was investigated in undifferentiated C2C12 skeletal muscle cells, after exposure to various apoptotic chemical triggers, chosen for their different mechanisms of action. Cells were pretreated with melatonin and then exposed to hydrogen peroxide, etoposide and staurosporine. Morphofunctional and molecular analyses show that in myoblasts melatonin prevents oxidative stress and apoptosis induced by chemicals following, at least in part, the mitochondria pathway. These results confirm melatonin ability to act as an antioxidant and antiapoptotic molecule in skeletal muscle cells, thus suggesting a possible therapeutic strategy for myopathies involving apoptosis misregulation. Microsc. Res. Tech. 79:532–540, 2016.

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.

Morphological adaptation and protein modulation of myotendinous junction following moderate aerobic training.

Myotendinous junction is the muscle-tendon interface through which the contractile force can be transferred from myofibrils to the tendon extracellular matrix. At the ultrastructural level, aerobic training can modify the distal myotendinous junction of rat gastrocnemius, increasing the contact area between tissues. The aim of this work is to investigate the correlation between morphological changes and protein modulation of the myotendinous junction following moderate training. For this reason, talin, vinculin and type IV collagen amount and spatial distribution were investigated by immunohistochemistry and confocal microscopy. The images were then digitally analyzed by evaluating fluorescence intensity. Morphometric analysis revealed a significant increased thickening of muscle basal lamina in the trained group (53.1 ± 0.4 nm) with respect to the control group (43.9 ± 0.3 nm), and morphological observation showed the presence of an electron-dense area in the exercised muscles, close to the myotendinous junction. Protein concentrations appeared significantly increased in the trained group (talin +22.2%; vinculin +22.8% and type IV collagen +11.8%) with respect to the control group. Therefore, our findings suggest that moderate aerobic training induces/causes morphological changes at the myotendinous junction, correlated to the synthesis of structural proteins of the muscular basal lamina and of the cytoskeleton.

Melatonin prevents mitochondrial dysfunctions and death in differentiated skeletal muscle cells

Oxidative stress increase induces cellular damage and apoptosis activation, a mechanism believed to represent a final common pathway correlated to sarcopenia and many skeletal muscle disorders. The goal of this study is to evaluate if melatonin, a ROS scavenger molecule, is able to counteract or modulate myotube death. Here, differentiated C2C12 skeletal muscle cells have been treated with melatonin before chemicals known to induce apoptotic death and oxidative stress, and its effect has been investigated by means of morpho‐functional analyses. Ultrastructural observations show melatonin protection against triggers by the reducing of membrane blebbing, chromatin condensation, myonuclei loss and in situ DNA cleavage. Moreover, melatonin is able to prevent mitochondrial dysfunctions which occur in myotubes exposed to the trigger alone. These findings demonstrate melatonin ability in preventing apoptotic cell death in skeletal muscle fibers in vitro, suggesting for this molecule a potential therapeutic role in the treatment of various muscle disorders.

Cytoprotective effects of melatonin in C2C12 skeletal muscle cells: a multiple technical approach

Melatonin 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

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.