Melatonin alleviates hydrogen peroxide induced oxidative damage in MC3T3-E1 cells and promotes osteogenesis by activating SIRT1.

Oxidative stress is an important contributor to the development of osteoporosis. Melatonin, an indoleamine secreted by the pineal gland, has antioxidant properties. This study aims to explore whether melatonin can promote bone formation and elucidate the mechanisms underlying this process. In this study, we used an in vitro hydrogen peroxide (H2O2)-induced oxidative stress model in MC3T3-E1 cells and an in vivo ovariectomized osteoporotic bone defect model in rats to explore the protective effects of melatonin against osteoporotic bone defects along with the mechanism underlying these effects. We found that melatonin significantly increased alkaline phosphatase activity, mineralization capacity, and the expression of BMP2, RUNX2, and OPN in MC3T3-E1 cells treated with H2O2. Furthermore, melatonin was found to activate SIRT1, SIRT3 and inhibit p66Shc, reduce the intracellular reactive oxygen species levels, stabilize mitochondria, reduce malondialdehyde levels, increase superoxide dismutase activity, and reduce apoptosis in MC3T3-E1 cells treated with H2O2. Intriguingly, these effects could be reversed by the SIRT1 inhibitor EX527. In vivo experiments confirmed that melatonin improves the microstructure and bone mineral density of the distal femoral bone trabecula and promotes bone formation. Meanwhile, melatonin activated SIRT1, inhibited p66Shc and increased SIRT3 expression. Taken together, our findings showed that melatonin can restrain oxidative damage in MC3T3-E1 cells and promote osteogenesis by activating SIRT1 which regulate the activity of SIRT3 and inhibit the expression of p66Shc, suggesting that melatonin could be a potential therapeutic agent for osteoporosis-related bone metabolic diseases.

Pro-oxidant vitamin C mechanistically exploits p66Shc/Rac1 GTPase pathway in inducing cytotoxicity.

P66Shc is the master regulator of oxidative stress whose pro-oxidant functioning is governed by ser36 phosphorylation. Phosphorylated p66Shc via Rac1 GTPase activation modulates ROS levels which in turn influence its pro-oxidative functions. Vitamin C at higher concentrations exhibits cytotoxic activity in various cancers, inducing ROS mediated cell death via pro-apoptotic mechanisms. Here we show a novel role of p66Shc in mediating pro-oxidant activity of vitamin C. Effect of vitamin C on the viability of breast cancer and normal cells was studied. High doses of vitamin C decreased viability of cancerous cells but not normal cells. Docking study displayed significant binding affinity of vitamin C with p66Shc PTB domain. Western blot results suggest that vitamin C not only enhances p66Shc expression but also induces its ser36 phosphorylation. Vitamin C at high doses was also found to activate Rac1, enhance ROS production and induce apoptosis. Interestingly, ser36 phosphorylation mutant transfection and pretreatment with antioxidant N-acetylcysteine results indicate that vitamin C induced Rac1 activation, ROS production and apoptosis is p66Shc ser36 phosphorylation dependent. Overall, results highlight that vitamin C mechanistically explores p66Shc/Rac1 pathway in inducing apoptosis and thus can pave a way to use this pathway as a potential therapeutic target in breast cancers.

p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke.

Airway epithelial mitochondrial injury plays a critical role in the pathogenesis of chronic obstructive pulmonary disease (COPD). The p66Shc adaptor protein is a newly recognized mediator of mitochondrial dysfunction. However, little is known about the effect of p66Shc on airway epithelial damage in the development of COPD. The aim of the present study is to investigate the roles of p66Shc and its upstream regulators in the mitochondrial injury of airway epithelial cells (Beas-2b) induced by cigarette smoke extract (CSE). Our present study revealed that CSE increased p66Shc expression and its mitochondrial translocation in concentration and time-dependent manners in airway epithelial cells. And p66Shc siRNA significantly attenuated mitochondrial dysfunction and cell injury when airway epithelial cells were stimulated with 7.5% CSE. The total and phosphorylated expression of PKCß and PKCδ was significantly increased associated with mitochondrial dysfunction and cell injury when airway epithelial cells were exposed to 7.5% CSE. The pretreatments with pharmacological inhibitors of PKCß and PKCδ could notably suppress p66Shc phosphorylation and its mitochondrial translocation and protect the mitochondria and cells against oxidative damage when airway epithelial cells were incubated with 7.5% CSE. These data suggest that a novel PKCß/δ-p66Shc signaling pathway may be involved in the pathogenesis of COPD and other oxidative stress-associated pulmonary diseases and provide a potential therapeutic target for these diseases.