Claudia Lumy Yano

Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

BackgroundPatients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue.MethodsIn this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells.ResultsWF decreased the viability of C2C12 myotubes, especially at concentrations of 20–25 μg.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations.ConclusionThese results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model.

Osteoblast Adhesion Dynamics : A Possible Role for ROS and LMW-PTP

Reactive oxygen species (ROS) modulate a variety of intracellular events, but their role in osteoblast adhesion and spreading remains unclear. ROS is a very‐known physiological modulators of Protein Tyrosine Phosphatases activities, mainly to low molecular weight protein tyrosine phosphatase (LMW‐PTP) activity. As this biological mechanism is not clear in osteoblast adhesion, we decided to investigate ROS levels and phosphorylations of FAK and Src, identifying these proteins as potential substrates to LMW‐PTP activity. Our results showed that during osteoblast adhesion/spreading (30 min and 2 h of seeding) the intracellular ROS content (hydrogen peroxide) is finely regulated by an effective anti‐oxidant system [catalase and Superoxide Dismutase (SOD) activities were evaluated]. During the first 30 min of adhesion, there was an increase in ROS production and a concomitant increase in focal adhesion kinase (FAK) activity after its phosphorylation at Tyrosine 397 (Y397). Moreover, after 2 h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW‐PTP expression at 30 min or 2 h. In order to validate our hypothesis that LMW‐PTP is able to control FAK activity by modulating its phosphorylation status, we decided to overexpress and silence LMW‐PTP in this context. Our results showed that FAK phosphorylation at Y397 was increased and decreased in osteoblasts with silenced or overexpressed LMW‐PTP, respectively. Together, these data show that ROS modulate FAK phosphorylation by an indirect way, suggesting that a LMW‐PTP/FAK supra‐molecular complex is involved in transient responses during osteoblast adhesion and spreading. J. Cell. Biochem. 115: 1063–1069, 2014.

Antioxidant defense and apoptotic effectors in ascorbic acid and β-glycerophosphate-induced osteoblastic differentiation

MC3T3‐E1 cells grown in the presence of ascorbic acid and β‐glycerophosphate (AA/β‐GP) express alkaline phosphatase and produce an extensive collagenous extracellular matrix. Differentiated MC3T3‐E1 cells are more sensitive to hydrogen peroxide‐induced oxidative stress than undifferentiated cells. In this study, we compared the profile of antioxidant enzymes and molecular markers of apoptosis in undifferentiated and differentiated MC3T3‐E1 cells (cell differentiation was induced by treatment with AA/β‐GP). Differentiated osteoblasts showed lower expression and activity of catalase, glutathione S‐transferase and glutathione peroxidase. The total superoxide dismutase activity and the expression of Cu/Zn superoxide dismutase were also lower, while the expression of Mn superoxide dismutase was higher in differentiated osteoblasts. The level of malondialdehyde, a widely used marker for oxidative stress, was lower in the AA/β‐GP group compared with control cells, but this difference was not significant. Western blotting showed that treatment with AA/β‐GP increased the Bax/Bcl‐2 ratio used as an index of cellular vulnerability to apoptosis. In addition, the activities of caspases 3, 8 and 9 and cleaved poly (ADP) ribose polymerase were significantly higher in differentiated cells. These findings provide new insights into how changes in the activities of major antioxidant enzymes and in the signaling pathways associated with apoptosis may influence the susceptibility of bone cells to oxidative stress.

Riboflavin and photoproducts in MC3T3-E1 differentiation.

Photoderivatives of riboflavin can modulate the proliferation and survival of cancer cells. In this work, we examined the influence of riboflavin and photoderivatives on osteoblast differentiation induced by ascorbic acid and β-glycerophosphate. These compounds decreased the osteoblast proliferation, increased the alkaline phosphatase activity, promoted a reduction in matrix metalloproteinase-2 activity and the decreased in the OPG/RANKL ratio. The effects of flavins on osteoblasts were unrelated to the antioxidant activity of these compounds. The biological activity of osteogenic medium containing riboflavin and its photoderivatives involved the activation of different signaling pathways (AKT, FAK, CaMKII), caspases-3, -8 and -9, and up-regulation of the expression and/or stabilization of osteoblastic transcription factors (Runx2 and β-catenin). These findings suggest a potential use of flavins as adjuvants to improve bone metabolism.

Osteoblast Adhesion Dynamics: A Possible Role for ROS and LMW-PTP: LMW-PTP AND ROS SIGNALING IN OSTEOBLAST ADHESION

Reactive oxygen species (ROS) modulate a variety of intracellular events, but their role in osteoblast adhesion and spreading remains unclear. ROS is a very‐known physiological modulators of Protein Tyrosine Phosphatases activities, mainly to low molecular weight protein tyrosine phosphatase (LMW‐PTP) activity. As this biological mechanism is not clear in osteoblast adhesion, we decided to investigate ROS levels and phosphorylations of FAK and Src, identifying these proteins as potential substrates to LMW‐PTP activity. Our results showed that during osteoblast adhesion/spreading (30 min and 2 h of seeding) the intracellular ROS content (hydrogen peroxide) is finely regulated by an effective anti‐oxidant system [catalase and Superoxide Dismutase (SOD) activities were evaluated]. During the first 30 min of adhesion, there was an increase in ROS production and a concomitant increase in focal adhesion kinase (FAK) activity after its phosphorylation at Tyrosine 397 (Y397). Moreover, after 2 h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW‐PTP expression at 30 min or 2 h. In order to validate our hypothesis that LMW‐PTP is able to control FAK activity by modulating its phosphorylation status, we decided to overexpress and silence LMW‐PTP in this context. Our results showed that FAK phosphorylation at Y397 was increased and decreased in osteoblasts with silenced or overexpressed LMW‐PTP, respectively. Together, these data show that ROS modulate FAK phosphorylation by an indirect way, suggesting that a LMW‐PTP/FAK supra‐molecular complex is involved in transient responses during osteoblast adhesion and spreading. J. Cell. Biochem. 115: 1063–1069, 2014.

Osteoblast Adhesion Dynamics

Reactive oxygen species (ROS) modulate a variety of intracellular events, but their role in osteoblast adhesion and spreading remains unclear. ROS is a very‐known physiological modulators of Protein Tyrosine Phosphatases activities, mainly to low molecular weight protein tyrosine phosphatase (LMW‐PTP) activity. As this biological mechanism is not clear in osteoblast adhesion, we decided to investigate ROS levels and phosphorylations of FAK and Src, identifying these proteins as potential substrates to LMW‐PTP activity. Our results showed that during osteoblast adhesion/spreading (30 min and 2 h of seeding) the intracellular ROS content (hydrogen peroxide) is finely regulated by an effective anti‐oxidant system [catalase and Superoxide Dismutase (SOD) activities were evaluated]. During the first 30 min of adhesion, there was an increase in ROS production and a concomitant increase in focal adhesion kinase (FAK) activity after its phosphorylation at Tyrosine 397 (Y397). Moreover, after 2 h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW‐PTP expression at 30 min or 2 h. In order to validate our hypothesis that LMW‐PTP is able to control FAK activity by modulating its phosphorylation status, we decided to overexpress and silence LMW‐PTP in this context. Our results showed that FAK phosphorylation at Y397 was increased and decreased in osteoblasts with silenced or overexpressed LMW‐PTP, respectively. Together, these data show that ROS modulate FAK phosphorylation by an indirect way, suggesting that a LMW‐PTP/FAK supra‐molecular complex is involved in transient responses during osteoblast adhesion and spreading. J. Cell. Biochem. 115: 1063–1069, 2014.