p66Shc Expression Research Articles

Carnosic acid attenuates neuropathic pain in rat through the activation of spinal sirtuin1 and down-regulation of p66shc expression

BackgroundIt has been reported that carnosic acid (CA) exhibits a range of biological activities including hepatoprotective, antioxidant and anti-inflammatory. However, the effect of carnosic acid in neuropathic pain remained elusive. MethodsA neuropathic pain model of chronic constriction injury (CCI) was established in adult male Sprague–Dawley rats. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded, and western blot was performed to detect sirtuin1 and p66shc content. ResultsIntrathecal administration of carnosic acid attenuated mechanical allodynia and thermal hyperalgesia in rats following chronic constriction injury. Interestingly, carnosic acid analgesic effect was positively associated with spinal sirtuin1 activation; however, p66shc was inhibited by carnosic acid in the spinal cord. In additional, sirtuin1 inhibitor EX-527 reversed the anti-nociceptive effect of carnosic acid. ConclusionsCarnosic acid is effective in the treatment of the established CCI-induced pain. It may be possible that spinal sirtuin1 activition by carnosic acid attenuates neuropathic pain through a mechanism involving the down-regulation of p66shc expression.

MiR214-regulated p53-NOX4/p66shc pathway plays a crucial role in the protective effect of Ginkgolide B against cisplatin-induced cytotoxicity in HEI-OC1 cells

The chemotherapeutic agent, cisplatin, is widely used for the treatment of several neoplastic diseases. The concomitant cytotoxicity in cochlear cells severely limits the maximum dose of cisplatin. Our previous study has shown that Ginkgolide B (GB) could protect against cisplatin-induced ototoxicity. In the present study, we aimed to elucidate the probable mechanism underlying GB-mediated protective effects against cisplatin-induced cytotoxicity. The results showed that, in HEI-OC1 auditory cells, both NOX4 and p66shc expression was increased by cisplatin. GB significantly reduced NOX4 and p66shc expression and superoxide generation. Over-expression of NOX4 or p66shc suppressed the inhibitory effects of GB on superoxide generation and the protective effects of GB on loss of cell viability and apoptosis associated with cisplatin. Moreover, p53 expression was increased by cisplatin. GB significantly decreased p53 expression and p53-binding of the promoters of NOX4 and p66shc. Over-expression of p53 suppressed the inhibitory effects of GB on NOX4 and p66shc expression and superoxide generation and the protective effects of GB on loss of cell viability and apoptosis associated with cisplatin. Furthermore, miR214 expression was decreased by cisplatin. GB significantly increased miR214 expression and inhibition of miR214 suppressed the inhibitory effects of GB on p53, NOX4 and p66shc expression and superoxide generation and the protective effects of GB against cisplatin-induced cytotoxicity. We demonstrate that GB decreases superoxide generation and the subsequent apoptosis through reduction of p53-mediated NOX4/p66shc pathway via up-regulation of miR214, resulting in attenuation of cisplatin-induced cytotoxicity. Our findings have gained an insight into the molecular mechanism of GB-exhibited inhibitory effect on cisplatin-induced cytotoxicity.

Diabetes-Induced Oxidative Stress in Endothelial Progenitor Cells May Be Sustained by a Positive Feedback Loop Involving High Mobility Group Box-1.

Oxidative stress is considered to be a critical factor in diabetes-induced endothelial progenitor cell (EPC) dysfunction, although the underlying mechanisms are not fully understood. In this study, we investigated the role of high mobility group box-1 (HMGB-1) in diabetes-induced oxidative stress. HMGB-1 was upregulated in both serum and bone marrow-derived monocytes from diabetic mice compared with control mice. In vitro, advanced glycation end productions (AGEs) induced, expression of HMGB-1 in EPCs and in cell culture supernatants in a dose-dependent manner. However, inhibition of oxidative stress with N-acetylcysteine (NAC) partially inhibited the induction of HMGB-1 induced by AGEs. Furthermore, p66shc expression in EPCs induced by AGEs was abrogated by incubation with glycyrrhizin (Gly), while increased superoxide dismutase (SOD) activity in cell culture supernatants was observed in the Gly treated group. Thus, HMGB-1 may play an important role in diabetes-induced oxidative stress in EPCs via a positive feedback loop involving the AGE/reactive oxygen species/HMGB-1 pathway.

Effect of testosterone on the regulation of p53 and p66Shc during oxidative stress damage in C2C12 cells

Accumulating evidence indicates that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H2O2)-induced apoptosis in C2C12 muscle cells, at different levels: morphological, physiological, biochemical and molecular. In the present study we observed that H2O2 induces the mitochondrial permeability transition pore (mPTP) opening and exerts p53 activation in a time-dependent way, with a maximum response after 1–2h of treatment. Testosterone treatment, prior to H2O2, reduces not only p53 phosphorylation but also p66Shc expression, activation and its mitochondrial localization, at the same time that it prevents the mPTP opening. Furthermore, testosterone diminishes JNK and PKCβI phosphorylation induced by H2O2 and probably contributing thus, to reduce the activation of p66Shc. Thus, the mPTP opening, p53, JNK and PKCβI activation, as well as p66Shc mRNA increase, induced by oxidative stress, were reduced by testosterone pretreatment. The data presented in this work show some of the components upstream of the classical apoptotic pathway, that are activated during oxidative stress and that are points where testosterone exerts its protective action against apoptosis, exposing some of the puzzle pieces of the intricate network that aged skeletal muscle apoptosis represents.

Small molecule compounds alleviate anisomycin-induced oxidative stress injury in SH-SY5Y cells via downregulation of p66shc and Aβ1-42 expression.

Oxidative stress and ageing are important factors contributing to the pathogenesis of Alzheimer's disease (AD), which is associated with neuronal damage and β-amyloid (Aβ) deposition. The p66shc adaptor protein is important for the regulation of oxidative stress and ageing. In the present study, SH-SY5Y human neuroblastoma cells were treated with anisomycin in order to establish a cell model of oxidative stress-induced neuronal damage. The results from quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and western blotting demonstrated that anisomycin was able to stimulate the secretion of Aβ1-42 from SH-SY5Y cells and upregulate the expression levels of p66shc, which was associated with concomitant damage to SH-SY5Y cells. In addition, the protective effects of various small molecule compounds with antioxidant properties against neuronal damage were evaluated. Notably, treatment of SH-SY5Y cells with gallic acid was associated with significant downregulation of p66shc protein expression levels, reduced anisomycin-induced secretion of Aβ1-42, and increased superoxide dismutase activity and acetylcholine secretion levels. The results of the present study suggested that anisomycin is able to promote oxidative neuronal damage by inducing the secretion of Aβ1-42 from neurons and increasing the neuronal expression of p66shc, and this damage may be attenuated by treatment with gallic acid. Therefore, gallic acid and similar small molecule compounds may be considered for the alleviation of neuronal oxidative stress injury in patients with AD.

Functions of the adaptor protein p66Shc in solid tumors

p66Shc is a 66 kDa Src homology 2 domain containing (Shc) adaptor protein homolog. Previous studies have demonstrated that p66Shc is a proapoptotic protein involved in the cellular response to oxidative stress and in regulating mammalian lifespan. However, accumulating evidence also indicates its critical role in solid tumor progression. The expression of p66Shc varies in different types of solid tumors, and it can paradoxically promote as well as suppress tumor progression, survival, and metastasis, depending on the cellular context. In this review, we discuss its functions in various solid tumors, the mechanisms by which it mediates the process of anoikis (detachment-induced cell death), and the epigenetic mechanisms that regulate its expression. These studies indicate the potential of p66Shc as a novel prognostic marker and therapeutic target for the prevention of tumor progression and metastasis.

Folic acid protects against arsenic-mediated embryo toxicity by up-regulating the expression of Dvr1.

As a nutritional factor, folic acid can prevent cardiac and neural defects during embryo development. Our previous study showed that arsenic impairs embryo development by down-regulating Dvr1/GDF1 expression in zebrafish. Here, we investigated whether folic acid could protect against arsenic-mediated embryo toxicity. We found that folic acid supplementation increases hatching and survival rates, decreases malformation rate and ameliorates abnormal cardiac and neural development of zebrafish embryos exposed to arsenite. Both real-time PCR analysis and whole in-mount hybridization showed that folic acid significantly rescued the decrease in Dvr1 expression caused by arsenite. Subsequently, our data demonstrated that arsenite significantly decreased cell viability and GDF1 mRNA and protein levels in HEK293ET cells, while folic acid reversed these effects. Folic acid attenuated the increase in subcellular reactive oxygen species (ROS) levels and oxidative adaptor p66Shc protein expression in parallel with the changes in GDF1 expression and cell viability. P66Shc knockdown significantly inhibited the production of ROS and the down-regulation of GDF1 induced by arsenite. Our data demonstrated that folic acid supplementation protected against arsenic-mediated embryo toxicity by up-regulating the expression of Dvr1/GDF1, and folic acid enhanced the expression of GDF1 by decreasing p66Shc expression and subcellular ROS levels.

Carnosic acid attenuates acute ethanol-induced liver injury via a SIRT1/p66Shc-mediated mitochondrial pathway.

Ethanol-induced liver injury is associated with oxidative stress and hepatocyte apoptosis. We previously demonstrated that SIRT1/p66Shc pathway activation attenuates hepatocyte apoptosis in liver ischemia/reperfusion. The current study aimed to investigate whether carnosic acid (CA), a natural antioxidant, can inhibit acute ethanol-induced apoptosis of hepatocytes and to determine the effect of SIRT1/p66Shc on this process. Our results showed that CA pretreatment significantly reduced ethanol-induced histologic damage, serum aminotransferase activity, and oxidative stress in rats. Importantly, CA pretreatment increased SIRT1 expression following ethanol exposure. Furthermore, p66Shc expression was negatively correlated with SIRT1 expression. Consistent with the results demonstrating p66Shc inhibition, CA pretreatment inhibited the release of cytochrome C and apoptosis-inducing factor (AIF) from mitochondria. After exposing L02 cells to ethanol, the increased SIRT1 expression induced by CA was abrogated by pharmacologic SIRT1 inhibition or the use of siRNA against SIRT1. Additionally, SIRT1 inhibition significantly abrogated the suppression of p66Shc expression and mitochondrial translocation induced by CA. Accordingly, CA-induced decreases in the release of cytochrome C and AIF and in mitochondrial apoptosis were nearly abolished by SIRT1 knockdown. These data indicated that CA-activated SIRT1 is protective against ethanol treatment. In summary, CA attenuates acute ethanol-induced liver injury via a SIRT1/p66Shc-mediated mitochondrial pathway.

The p66Shc protein controls redox signaling and oxidation-dependent DNA damage in human liver cells.

The p66Shc protein mediates oxidative stress-related injury in multiple tissues. Steatohepatitis is characterized by enhanced oxidative stress-mediated cell damage. The role of p66Shc in redox signaling was investigated in human liver cells and alcoholic steatohepatitis. HepG2 cells with overexpression of wild-type or mutant p66Shc, with Ser36 replacement by Ala, were obtained through infection with recombinant adenoviruses. Reactive oxygen species and oxidation-dependent DNA damage were assessed by measuring dihydroethidium oxidation and 8-hydroxy-2'-deoxyguanosine accumulation into DNA, respectively. mRNA and protein levels of signaling intermediates were evaluated in HepG2 cells and liver biopsies from control and alcoholic steatohepatitis subjects. Exposure to H2O2 increased reactive oxygen species and phosphorylation of p66Shc on Ser36 in HepG2 cells. Overexpression of p66Shc promoted reactive oxygen species synthesis and oxidation-dependent DNA damage, which were further enhanced by H2O2. p66Shc activation also resulted in increased Erk-1/2, Akt, and FoxO3a phosphorylation. Blocking of Erk-1/2 activation inhibited p66Shc phosphorylation on Ser36. Increased p66Shc expression was associated with reduced mRNA levels of antioxidant molecules, such as NF-E2-related factor 2 and its target genes. In contrast, overexpression of the phosphorylation defective p66Shc Ala36 mutant inhibited p66Shc signaling, enhanced antioxidant genes, and suppressed reactive oxygen species and oxidation-dependent DNA damage. Increased p66Shc protein levels and Akt phosphorylation were observed in liver biopsies from alcoholic steatohepatitis compared with control subjects. In human alcoholic steatohepatitis, increased hepatocyte p66Shc protein levels may enhance susceptibility to DNA damage by oxidative stress by promoting reactive oxygen species synthesis and repressing antioxidant pathways.

Palmitic acid increases pro-oxidant adaptor protein p66Shc expression and affects vascularization factors in angiogenic mononuclear cells: Action of resveratrol

A defect in neo-vascularization process involving circulating angiogenic mononuclear cells (CACs) dysfunction is associated with diabetes. We showed that oxidative stress was elevated in CACs cultured from blood of individuals with metabolic syndrome (MetS) and diabetes. We then assessed the action of palmitic acid (PA), a deregulated and increased NEFA in metabolic disorders, focusing on its oxidant potential. We observed that the phyto-polyphenol resveratrol normalized oxidative stress both in CACs isolated from MetS patients or treated with PA. Resveratrol further decreased the deleterious action of PA on gene expression of vascularization factors (TNFα, VEGF-A, SDF1α, PECAM-1, VEGFR2, Tie2 and CXCR4) and improved CAC motility. Particularly, resveratrol abolished the PA-induced over-expression of the pro-oxidant protein p66Shc. Neither KLF2 nor SIRT1, previously shown in resveratrol and p66Shc action, was directly involved. Silencing p66Shc normalized PA action on VEGF-A and TNFα specifically, without abolishing the PA-induced oxidative stress, which suggests a deleterious role of p66Shc independently of any major modulation of the cellular oxidative status in a high NEFA levels context. Besides showing that resveratrol reverses PA-induced harmful effects on human CAC function, certainly through profound cellular modifications, we establish p66Shc as a major therapeutic target in metabolic disorders, independent from glycemic control.

Post-ischaemic silencing of p66Shc reduces ischaemia/reperfusion brain injury and its expression correlates to clinical outcome in stroke.

Constitutive genetic deletion of the adaptor protein p66(Shc) was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66(Shc) gene regulation in human ischaemic stroke. Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66(Shc) was injected intravenously. We observed that post-ischaemic p66(Shc) knockdown preserved blood-brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66(Shc) preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66(Shc) gene expression is transiently increased and that this increase correlates with short-term neurological outcome. Post-ischaemic silencing of p66(Shc) upon reperfusion improves stroke outcome in mice while the expression of p66(Shc) gene correlates with short-term outcome in patients with ischaemic stroke.

P53 Contributes to Cisplatin Induced Renal Oxidative Damage via Regulating P66shc and MnSOD

Background/Aims: Cisplatin is widely used to treat malignancies. However, its major limitation is the development of dose-dependent nephrotoxicity. The precise mechanisms of cisplatin-induced kidney damage remain unclear. Previous study demonstrated the central role of mitochondrial ROS (mtROS) in the pathogenesis of cisplatin nephrotoxicity. The purpose of this study was to explore the mechanism of mtROS regulation in cisplatin nephrotoxicity. Methods: p53, MnSOD and p66shc were detected at mRNA and protein levels by qPCR and western blot in HK2 cells. mtROS levels were determined by DCFDA and MitoSOX staining. Cell viability and cell apoptosis were accessed by CCK-8 assay, TUNEL assay and flow cytometry, respectivesly. siRNAs were used to knock down p53 and p66shc expression and subsequent changes were observed. In vivo assays using a mouse model of cisplatin-induced acute kidney injury were used to validate the in vitro results. Results: In HK2 cells, cisplatin exposure decreased the MnSOD and increased the expression of p53 and p66shc. MnTBAP, a MnSOD mimic, blocked cisplatin-induced the generation of mtROS and cell injury. P66shc and p53 siRNAs rendered renal cells resistant to cisplatin-induced mtROS production and cell death. Furthermore, knockdown of p53 restored MnSOD and inhibiting p66shc. Consistent with these results, we revealed that p53 inhibitor reduced cisplatin-induced oxidative stress and apoptosis by regulating MnSOD and p66shc in the kidney of cisplatin-treated mice. Conclusion: Our study identifies activation of p53 signalling as a potential strategy for reducing the nephrotoxicity associated with cisplatin treatments and, as a result, broadens the therapeutic window of this chemotherapeutic agent.

Nafamostat Mesilate Inhibits TNF-α-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production.

Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-α (TNF-α). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-α for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 µg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-α (3 ng/mL), and it dose dependently prevented the TNF-α-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-α-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-α-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.

Abstract 14746: Dynamic SIRTUIN1-modulated Lysine Acetylation of P66shc Governs Vascular Endothelial Oxidative Stress and Dysfunction of Diabetes

The SIRTUIN1 lysine deacetylase (SIRT1) ameliorates diabetic vascular dysfunction by epigenetically suppressing endothelial expression of the oxidative stress protein p66shc. However, whether SIRT1 modulates the oxidative function of p66shc by directly targeting it for lysine deacetylation is not known. Here we show that the oxidative function of p66shc is dynamically modulated by lysine acetylation. Mass spectroscopy identified lysine 81 in the unique CH2 domain of p66shc as the residue targeted by SIRT1. High glucose and SIRT1 knockdown stimulates acetylation of lysine 81 of endothelial p66shc. Compared to WT p66shc, non-acetylatable (K81R) p66shc is significantly handicapped in promoting endothelial hydrogen peroxide production stimulated by high glucose. Compared to WT p66shc, K81R p66shc is also less prone to serine 36 phosphorylation by high glucose, which is essential for the oxidative function of p66shc. Moreover, in contrast to WT p66shc which worsens endothelial dysfunction, expression of K81R p66shc does not impair endothelial function in wild type mice and rescues endothelium dysfunction of diabetic db+/db+ mouse aortas . These findings show that lysine 81 acetylation promotes the oxidative role of p66shc in hyperglycemic conditions, and is essential for p66shc-mediated endothelial dysfunction.

The cellular prion protein counteracts cardiac oxidative stress.

The cellular prion protein, PrP(C), whose aberrant isoforms are related to prion diseases of humans and animals, has a still obscure physiological function. Having observed an increased expression of PrP(C) in two in vivo paradigms of heart remodelling, we focused on isolated mouse hearts to ascertain the capacity of PrP(C) to antagonize oxidative damage induced by ischaemic and non-ischaemic protocols. Hearts isolated from mice expressing PrP(C) in variable amounts were subjected to different and complementary oxidative perfusion protocols. Accumulation of reactive oxygen species, oxidation of myofibrillar proteins, and cell death were evaluated. We found that overexpressed PrP(C) reduced oxidative stress and cell death caused by post-ischaemic reperfusion. Conversely, deletion of PrP(C) increased oxidative stress during both ischaemic preconditioning and perfusion (15 min) with H2O2. Supporting its relation with intracellular systems involved in oxidative stress, PrP(C) was found to influence the activity of catalase and, for the first time, the expression of p66(Shc), a protein implicated in oxidative stress-mediated cell death. Our data demonstrate that PrP(C) contributes to the cardiac mechanisms antagonizing oxidative insults.

Role of adapter protein p66Shc in injured mediating alveolar epithelial cells induced by hyperoxia

Objective To determine the expression of adapter protein p66Shc in mediating alveolar epithelial cells induced by hyperoxia and to explore their relationship. Methods A549 cells were cultured in vitro and divided randomly into a control group and a hyperoxia group.The hyperoxia group was exposed to a mixture of oxygen(O2, 900 mL /L) and carbon dioxide(CO2, 50 mL /L) for 10 min, then cultured in a closed environment.The changes in morphology were observed under inverted microscope after exposure to oxygen or air for 24 hours.The cell apoptosis was detected by flow cytometry(FC) after 24 hours.And the expression of p66Shc was detected by immunohistochemical method after 24 hours.The correlation of the changes in mitochondrial membrane potential and p66Shc protein expression was analyzed by using Bivariate correlation analysis. Results 1.Under inverted microscopy, A549 cells from the air group significantly increased, stuck to each other tightly and grew very quickly.Their adhesion was better, multy-angle oblate and many cells were in division phase.Compared with the control group, the changes in morphology of A549 were remarked and obvious than those in the hyperoxia group.The cells grew slowly, their counts decreased and the cell morphology changed from typical multi-angle oblate to round or ellipse.2.Compared with the control group, after 24 h, in hyperoxia group of A549 cells, red fluorescence decreased, and green fluorescence enhanced.3.Compared with the controls(0.057 664 88±0.006 517 84), the expression of p66Shc(0.123 600 50±0.004 227 23) was significantly increased in the hyperoxia group(t=-24.006, P<0.001).4.The decline of membrane potential was negatively correlated with the increased expression of p66Shc protein(R=-0.988, P<0.001). Conclusions The hyperoxia induction could significantly increase in injured mediating alveolar epithelial cells induced by hyperoxia, the expression of p66Shc increases, the membrane potential declined, and they exhibit a negative correlation.So p66Shc may be involved in the process of high oxygen damage to human alveolar epithelial cells. Key words: Hyperoxia; Alveolar epithelial cells; Adapter protein; Oxidative stress; Apoptosis

P66Shc mediates increased platelet activation and aggregation in hypercholesterolemia

Background and hypothesisHypercholesterolemia leads to a prothrombotic phenotype. Platelet hyperactivity associated with hypercholesterolemia has been attributed, in part, to oxidative stress. P66Shc is a well-known determinant of cellular and organismal oxidative stress. However, its role in platelet biology is not known. We hypothesized that p66Shc mediates platelet hyperactivation and hyperaggregation in hypercholesterolemia. Methods and resultsP66Shc was expressed in both human and mouse platelets, as determined by qRT-PCR and immunoblotting. Mouse platelet p66Shc expression was upregulated by hypercholesterolemia induced by high-fat diet feeding. Compared to wild-type mice, high-fat diet-induced p66Shc expression in platelets was suppressed in transgenic mice expressing a short hairpin RNA targeting p66Shc (p66ShcRNAi). High-fat diet feeding of wild-type mice amplified surface P-selectin expression on platelets stimulated by the thrombin receptor agonist protease-activated receptor-4 (PAR4), and increased aggregation of platelets induced by thrombin. These exaggerated platelet responses induced by high-fat diet feeding were significantly blunted in p66ShcRNAi mice. Finally, thrombin-stimulated platelet reactive oxygen species were suppressed in p66ShcRNAi mice. ConclusionsHypercholesterolemia stimulates p66Shc expression in platelets, promoting platelet oxidative stress, hyperreactivity and hyperaggregation via p66Shc.

Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene.

The mammalian Shc family, composed of p46, p52, and p66 isoforms, serves as an adaptor protein in cell growth and stress response. p66Shc was shown to be a negative lifespan regulator by acting as a prooxidant protein in mitochondria; however, the regulatory mechanisms of p66Shc expression and function are incompletely understood. This study provides evidence for new features of p66Shc serving as an antioxidant and critical protein in cell differentiation. Unique among the Shc family, transcription of p66Shc is activated through the antioxidant response element (ARE)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in K562 human erythroleukemia and other cell types after treatment with hemin, an iron-containing porphyrin. Phosphorylated p66Shc at Ser-36, previously reported to be prone to mitochondrial localization, is increased by hemin treatment, but p66Shc remains exclusively in the cytoplasm. p66Shc knockdown inhibits hemin-induced erythroid differentiation, in which reactive oxygen species production and apoptosis are significantly enhanced in conjunction with suppression of other ARE-dependent antioxidant genes. Conversely, p66Shc overexpression is sufficient for inducing erythroid differentiation. Collectively these results demonstrate the isoform-specific regulation of the Shc gene by the Nrf2-ARE pathway and a new antioxidant role of p66Shc in the cytoplasm. Thus p66Shc is a bifunctional protein involved in cellular oxidative stress response and differentiation.

The expression of p66shc in peripheral blood monocytes is increased in patients with coronary heart disease and correlated with endothelium-dependent vasodilatation

The objective of this study is to detect the p66shc mRNA and protein expression of the peripheral blood monocytes (PBMs) in coronary heart disease patients (CHD) and controls, to evaluate the correlation between the expression of p66shc mRNA in the PBMs and endothelium-dependent vasodilatation. This study included 78 coronary angiography-documented CHD patients (CHD group) and 38 non-CHD controls (control group). The p66shc mRNA and protein levels were determined by quantitative real-time PCR and western blotting. The flow-mediated dilatation (FMD, endothelium-dependent), nitroglycerine-induced dilatation (NID, endothelium-independent) and carotid intimal medial thickness (CIMT) were detected using high-resolution ultrasound. The p66shc mRNA and the protein expression levels in the PBMs were significantly higher in the CHD group compared with the control group (p=0.007 and 0.001). The FMD (p<0.001) and NID (p=0.013) were significantly lower and the CIMT (p=0.007) was significantly thicker in the CHD patients than in the controls. In the univariate analysis, the expression of the p66shc mRNA in the PBMs was significantly positively correlated with the serum LDL-C and homocysteine levels and the CIMT and was inversely correlated with the FMD and the NID (all p<0.001). In the multiple linear regression analysis, the FMD (p<0.001), LDL-C (p=0.002) and homocysteine levels (p=0.002) remained independently correlated with the p66shc mRNA expression. These findings highlight a pivotal role for the expression of p66shc in CHD and endothelial dysfunction, which might represent a molecular target to prevent endothelial dysfunction-related disease.

The Expression of p66Shc Protein in Benign, Premalignant, and Malignant Gastrointestinal Lesions

ROS produced from Oxidative stress have long been recognized to be involved in carcinogenesis. p66Shc generates H(2)O(2) by oxidizing cytochrome c, and its expression has been reported to be elevated in several tumors. However, the expression of p66Shc in gastric cancer has not been reported, and its role in colorectal cancer has not been well elucidated. This study investigated p66Shc expression in benign, premalignant, and malignant gastric and colorectal lesions. p66Shc expression in 146 gastric tumors, 136 colorectal tumors, 45 gastric hyperplastic polyps, 33 gastric low-grade intraepithelial neoplasias, 41 gastric high-grade intraepithelial neoplasias, 42 colorectal hyperplastic polyps, 21 colorectal low-grade intraepithelial neoplasias, 38 colorectal high-grade intraepithelial neoplasias, and 30 normal gastric and colorectal tissues was measured by immunohistochemistry. Most normal gastric and colorectal tissues exhibited low or no p66Shc expression (93.4 %), while most gastric and colorectal tumors exhibited moderate to high p66Shc expression (78.1 %-80.9 %). The p66Shc expression in normal gastric and colorectal tissues were significantly lower than that in the low-grade intraepithelial neoplasias (p < 0.05), high-grade intraepithelial neoplasias (p < 0.01), and gastric adenocarcinomas (p < 0.01 or <0.001). No differences in p66Shc expression were observed in gastric and colorectal hyperplastic polyps compared to the normal tissues. No statistically significant differences in p66Shc expression were observed between patients with different disease stages, different tumor grades, and with or without lymph node metastasis in gastric and colorectal cancers. In conclusion, p66Shc may be involved in the carcinogenesis of gastric and colorectal cancers and could be a marker for the diagnosis of gastric and colorectal cancers.