Recent experimental pieces of evidence demonstrate that PPARγ is implicated in the oxidative stress response, an imbalance between antithetic prooxidation and antioxidation forces that may lead the cell to apoptotic or necrotic death. In this delicate and intricate game of equilibrium, PPARγ stands out as a central player devoted to the quenching and containment of the damage and to foster cell survival. However, PPARγ does not act alone: indeed the nuclear receptor is at the point of interconnection of various pathways, such as the nuclear factor erythroid 2-related factor 2 (NRF2), Wnt/β-catenin, and forkhead box proteins O (FOXO) pathways. Here we reviewed the role of PPARγ in response to oxidative stress and its interaction with other signaling pathways implicated in this process, an interaction that emerged as a potential new therapeutic target for several oxidative-related diseases.Peroxisome-proliferator activator receptor  Reactive oxygen species (ROS) are free radicals and reactive metabolites containing oxygen with unpaired electron; potentially harmful, they act as potent oxidants for lipids, proteins, and DNA . Small aBecause high levels of ROS may be detrimental, the cells possess a vast array of antioxidant systems devoted to ROS neutralization and maintaining of a balance between prooxidants and antioxidants. We distinguish two types of antioxidants: enzymatic and nonenzymatic . Enzymatic antioxidants are proteins such as superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase, and catalase, while chemical antioxidants are scavenger molecules such as vitamin C and D and glutathione (GSH) . ProoxidWhen the near equilibrium condition between prooxidants and antioxidants is perturbed, the ensuing imbalance determines the so-called oxidative stress. During oxidative stress the sustained production of ROS and of other high reactive species, not sufficiently quenched by antioxidants, may deal significant damage to the cells, and, if too much damage is done, that may lead to necrotic or apoptotic cell death.Excessive ROS production and oxidative stress is believed to be the cause, or is linked to, the establishment of several pathologies (for a list see ), includα, PPARγ, and PPAR β/δ, also known as NR1C1, NR1C3, and NR1C2 [γ is the only receptor known to possess three splicing isoforms characterized by a different tissue distribution [The Peroxisome-proliferator activator receptors (PPARs) form a family of ligand-activated nuclear receptor transcription factors that regulates the function and expression of complex gene networks, especially involved in energy homeostasis and inflammation –17. The nd NR1C2 . Among tribution , 16.γ shares a protein structure common to the other PPARs and to most of the nuclear receptors characterized by 4 functional domains  [PPARand E/F) , 19. Theand E/F) , 16, 19.γ does not act alone but regulates genes transcription acting as heterodimer with the retinoid X receptor (RXR) [γ : RXR bind to PPAR response elements (PPREs) located in the promoter region of target genes. PPREs are hexanucleotide direct repeat consensus elements, whose sequence is AGGTCA, separated by one or two bases (known as Direct Repeat 1 (DR-1) and 2 (DR-2)).PPARor (RXR) , 16. As γ in a cell context depends on the presence of other molecules such as coactivators and corepressors, expression of other PPARs, availability of RXR, the status of promoter of the target genes, and presence of endogenous ligands [γ is canonically obtained through ligand binding. The ligand-binding determines a conformational change in the three-dimensional structure of PPARγ : RXR dimers that is accompanied by loss of heat shock proteins, detachment of corepressors, and recruitment of coactivators [γ have also been described [γ and RXR demonstrated that PPARγ nuclear distribution changes after the coexpression of RXR independently from the presence or absence of PPARγ- or RXR-specific ligands, suggesting that RXR is implicated in the nuclear reorganization of PPARγ.The activity of PPAR ligands . Activattivators , 16, 21.escribed . Akiyamaescribed  utilizinγ may also be modulated by posttranslational modifications, such as phosphorylations, independently from ligand binding [γ [γ. The activated receptor then binds to PPRE: the net results of this complex mechanism are changes in the expression level of mRNAs encoding PPARγ target genes.The activation status of PPAR binding , 23. Indnding [γ ; this evnding [γ , cytoplanding [γ , 25, andnding [γ  of PPARγγ may also influence genes expression with a mechanism known as transrepression. Transrepression does not require DNA binding of the activated receptor to the PPRE and has been invoked to explain anti-inflammatory action of PPARγ [PPARof PPARγ –30; seveof PPARγ .γ ligands [γ is implicated in oxidative stress response. This hypothesis is also corroborated by the sheer number of scientific papers published on this topic: in fact, a query for “PPARγ and oxidative stress” in PubMed returns (as to August 2011) at least 300 publications.The experimental evidence that oxidized lipids and 15-deoxy-delta -prostaglandin J(2) (15d-PGJ2), which are natural endogenous PPAR ligands , are proγ is emerging as an important regulator of the response to this condition, in this paper we will summarize the current understanding of the role of this nuclear receptor in response to oxidative stress and its interaction with other signaling pathways implicated in this process.Given that various pathologies are associated to oxidative stress and PPARγ-specific agonists. Together with other PPARγ agonists, they are used or are under studies for the treatment of oxidative stress-related diseases such as diabetes, vascular diseases [γ dependent, TZD PPARγ-independent effects have been widely described and may coexist with the PPARγ-dependent effects [γ, via inhibition of nucleophosmin that is associated with the induction of p53 phosphorylation and p21 expression [γ, decreases ROS generation induced by high glucose treatment, whereas it induces the antioxidant enzyme heme-oxygenase 1 (HO-1) in a PPARγ-dependent manner [The insulin-sensitizing drugs thiazolidinediones (TZDs), used in the treatment of type II diabetes, are PPARdiseases –35, Parkdiseases , Alzheimdiseases , nonalcodiseases , and Hundiseases . It is w effects –44. As epression ; in QZG t manner .γ may directly modulate the expression of several antioxidant and prooxidant genes in response to oxidative stress  [Furthermore, ligand-activated PPAR (MnSOD) , 51, GPx (MnSOD) , the sca (MnSOD) , 54, end (MnSOD) , HO-1 [4 (MnSOD) , 55, 56, (MnSOD) , 38, whe (MnSOD) , 57–59.2− to O2 and H2O2, is increased by rosiglitazone [γ knockout mice MnSOD is downregulated at the transcriptional and translational levels with a consequent increase of O2− levels [γ [The activity of mitochondrial MnSOD, which oversees the dismutation of Olitazone ; moreove− levels . In factevels [γ .2O2 to H2O and O2  and acting as scavenging for oxidized lipids. Recently, Chung et al. [γ induces GPx3 and protects from oxidative stress.GPx protect cells from oxidative stress in two ways, reducing Hg et al.  demonstrγ [2− in the mitochondria and facilitating the transport of mitochondrial ROS to the cytosol, where they would stimulate the expression of neuroprotective genes  [In spontaneous hypertensive rats, oral intake of rosiglitazone upregulates UCP2 and the protective effects of rosiglitazone are abrogated silencing PPARγ . The mitd Bcl-2) .γ. In fact, it has been demonstrated that in murine macrophages CD36 expression increases after the treatment with PPARγ ligands [γ binding to functional active PPRE located in the gene promoter [The expression of the scavenger receptor CD36 that mediates the recognition and internalization of oxidized lipids  may also ligands , probablpromoter .γ modulates the expression of eNOS and iNOS [2− forming the highly reactive peroxynitrite. In mice with an endothelial specific knockout of PPARγ aortic segments release less nitric oxide than those from controls and the reduced expression correlates with an increase in the parameters of oxidative stress [γ protect from oxidative stress controlling eNOS expression. Conversely, the high production of NO by iNOS is usually associated to complex immunomodulatory and antitumoral mechanisms and dysfunctional induction of iNOS expression seems to be involved in the pathophysiology of several human diseases [γ agonists repress iNOS expression in various cells such as activated macrophages [γ from reactive peroxynitrite.PPARand iNOS , 33, 59.rophages , and liprophages  suggesti2−, contributing to oxidative stress. Besides, PGH2 also inhibits apoptosis, favors cell adhesion, motility, invasion, and promotes angiogenesis [γ acts on COX-2 expression is still debated. In fact during oxidative stress a PPARγ-mediated inhibition as well as induction of COX-2 has been reported [γ agonist pioglitazone and rosiglitazone protect the cells from oxidative stress after ischemia suppressing COX-2 expression in a PPARγ-dependent manner [γ-dependent manner. This latter result is in agreement with that of Aleshin et al. [γ-dependent pathway.COX-2 is an inducible form of ciclooxygenase that contributes to the metabolism of arachidonic acid forming prostaglandin H2 (PGH2), a precursor of 15d-PGJ2 , 8. The reported , 57, 58.t manner  demonstrn et al.  that desγ on the expression of HO-1. HO-1 can be strongly induced in many tissues in response to cellular stress caused by a wide spectrum of stimuli, including ROS and prostaglandins [γ induces HO-1 expression interacting with two PPRE DR-1 located between −1740 kb and −1826 kb from the transcription start site [γ increases the expression of HO-1 during oxidative stress induced by high glucose [γ agonist 15d-PGJ2 may induce HO-1 independently of PPARγ  via nuclear factor erythroid 2-related factor 2 (NRF2) [Still debated is also the effect of PPARglandins . Enzymatglandins . In humaart site . Moreove glucose  and in t glucose ; however glucose . To furt2 (NRF2)  or GSH-d2 (NRF2) .γ may also influence the antioxidant and anti-inflammatory responses interacting with other regulatory pathways. PPARγ has shown to induce anti-inflammatory responses inhibiting proinflammatory transcription factors such as nuclear factor Kappa-light-chain-enhancer of activated B cells (NF-κB) [κB factors are of central importance in inflammation and their role in response to ROS has been recently reviewed [κB action is usually proinflammatory and prooxidant inducing the expression of genes such as iNOS and COX-2, but it may also regulate the expression of SOD and other anti-inflammatory genes [γ may reduce NF-κB activities in various ways. PPARγ transrepresses NF-κB activation either by forming a repressor complex in the promoter of NF-κB-target genes or by direct binding with NF-κB [κB activation mediated by H2O2 [κB negatively regulates PPARγ transcriptional activity with a mechanism that requires the presence of histone deacetylase 3 (HDAC3) [κB could, potentially, also induce PPARγ via a thioredoxin- (TRX-) dependent mechanism. TRX is a potent antioxidant protein whose expression depends on NF-κB [γ, suggesting a link between TRX-TBP-2 and the nuclear receptor [PPAR (NF-κB) –30, 66  , 68. Inton NF-κB ; the acton NF-κB . TBP-2 nreceptor .γ modulates oxidative stress responses interacting with NRF2 and the Wnt/β-catenin and forkhead box proteins O (FOXO) pathways.Furthermore, new experimental pieces of evidence suggest that PPARThe nuclear factor erythroid 2-related factor 2 (NRF2), a redox-sensitive member of the cap “n” collar basic leucine zipper family, plays a vital role in cytoprotection against oxidative and electrophilic stress as well as in suppression of inflammation .κB signaling by targeting the inhibitor of NF-κB subunit β (IKKβ) [Expression of NRF2 is tightly regulated by the Kelch-like ECH-associated protein 1 (Keap1) , an E3 uβ (IKKβ) .In unstressed conditions, NRF2 remains in the cytosol and it is rapidly degraded by proteasome after Keap1-mediated ubiquitination. In presence of ROS or electrophilic agents, the degradation ceases and NRF2 translocates and accumulates in the nucleus where it regulates the transcription of genes containing in the promoter region the antioxidant response element (ARE). NRF2 nuclear translocation and transcriptional activity depend also on the phosphorylation status of the transcription factor , 73–75; The number of targets of NRF2 is continuously expanding and comprises NADPH-generating enzymes , glutathγ expression is compromised in NRF2 null mice, in normal and stress conditions [γ was markedly reduced compared to wild-type animals [γ, were generally downregulated compared with the wild-type mice [γ reduced expression is a direct effect of the lack of NRF2; in fact both Huang et al. [γ expression binding to at least two ARE sequences  in the upstream promoter region of the nuclear receptor.Recently, microarray analysis of NRF2 target genes demonstrated that PPARnditions –81. In t animals , 80, wheγ agonists exert potent anti-inflammatory activities; interestingly, rosiglitazone and other PPARγ ligands such as 15d-PGJ2 increase NRF2 expression dependently from PPARγ [γ levels and putative PPREs have been identified on NRF2 gene promoter [γ and NRF2, where PPARγ may act directly or through upstream pathways for NRF2 activation. This circuit may not only explain the effects of PPARγ agonists on NRF2 expression but may also contribute to the anti-inflammatory effects associated with PPARγ activation. Thimmulappa et al. [κB, increasing lung inflammation. In fact, NRF2 regulates NF-κB activation largely by modulating its MyD88-dependent and -independent upstream signaling components; the restrain of proinflammatory signaling pathways is apparently associated with the NRF2 ability to maintain redox homeostasis. In this sense, the PPARγ-mediated activation of NRF2 signaling, which results in NRF2-dependent inhibition of proinflammatory pathways, may strengthen, and contribute to, the anti-inflammatory activity of PPARγ.As previously stated, PPARom PPARγ , 77, 82.promoter ; this sua et al.  demonstrα [γ may increase oxidative stress that acts as “second hit” in NASH; in fact, on the basis of experimental evidence, we may hypothesize that NRF2, together with PPARγ, preserves from the progression of NASH throughout protective effects on oxidative stress, inflammation, and lipid metabolism.The altered expression of genes involved in inflammation and lipid metabolism in NRF2 null mice is interesting for the pathogenesis of NAFLD and its progression to NASH. Lack of NRF2 in mice exposed to high-fat or methionine-choline diet increases the oxidative stress-related damage and the lipid accumulation mediated by LXRα , 85. Theγ is not a simple downstream effector of the NRF2 pathway but may also act synergically with NRF2 in the activation of antioxidant genes [GSTA2 gene by PPARγ and RXR ligands, as evidenced by the binding site-deleted promoter-luciferase assay, and it is necessary for the full gene transactivation [γ and NRF2 may act independently to induce the transcription of the same gene, depending on the cellular context. In murine macrophage, oxidized lipids that in macrophages induce a PPARγ-dependent CD36 expression [γ-independent expression of the scavenger [What is certain is that PPARnt genes , 77. In tivation , 54. On pression , 54, in cavenger ; the latcavenger .β-catenin pathway is a well-known morphogenic signaling pathway first discovered in colon carcinoma [β-catenin has been described in several processes including morphogenesis, maintenance of stem cells characteristics but also cell differentiation, cell survival, and lipid and glucose metabolism [The Wnt/arcinoma . From thtabolism –91; in fβ-catenin-DNA binding protein T cell factor/lymphoid enhancer factor (TCF/LEF) [β-catenin in the so-called noncanonical pathway [via mTOR [The main effector of Wnt, in the canonical pathway, is the bipartite transcription TCF/LEF) ; Wnt may pathway , 93 and via mTOR .β-catenin is constitutively expressed in the cytoplasm, but its activity is regulated at the posttranscriptional level through phosphorylation. β-catenin phosphorilation is performed by a degradation complex comprised, among other proteins, of glycogen synthase kinase 3 beta (GSK3β) and adenomatous polyposis coli (APC) and results in ubiquitination and degradation by the proteasome.β phosphorylation followed by the stabilization and nuclear translocation of β-catenin, and hence activation of the pathway. To add further complexity to the system, other signaling pathways, such as the insulin and insulin-like growth factor 1 (IGF-1) pathways, activate β-catenin possibly through an AKT-dependent GSK3β phosphorylation [Conversely, the binding of Wnt glycoproteins to the seven pass membrane receptor Frizzled and coreceptors low-density lipoprotein receptor-related protein 5/6 (LRP5/6) results in GSK3rylation , 96.β-catenin is deeply involved in the response to oxidative stress. Products of lipid oxidation activate the canonical Wnt pathway in a rat model of diabetic retinopathy [β-catenin and TCF/LEF gene transcription in the bone [κB, may influence GSK3β phosphorylation: RKIP depletion increases ROS-induced p38 MAPK activation which inhibits GSK3β increasing β-catenin levels [β-catenin and induces cell-cycle arrest and apoptosis [β-catenin in the developing liver determines embryonic lethality at E17 associated with increased apoptosis and oxidative stress [β-catenin increases the oxidative stress [β-catenin may also be involved in the regulation of antioxidant enzymes such as GST [Recent experimental pieces of evidence suggest that the Wnt/inopathy  and Wnt inopathy ; conversthe bone , while nthe bone , 100. Thn levels . Furtherpoptosis . In the e stress ; these re stress . Interesh as GST  and cytoh as GST .β-catenin in the response to oxidative stress but also imply that its role is multifaceted. Interestingly, some of the variegated effects of the Wnt/β-catenin may be explained by the introduction of another player in this complex regulatory pathway: the FOXO proteins.The described experimental results not only demonstrate the involvement of the Wnt/β-catenin/TCF activation and plays an important role in stress response to ROS. In mammals, the FOXO family comprises four isoforms  characterized by an overlapping expression during development and in adulthood. FOXO acts as transcriptional activators governing a variety of different cellular processes in response to different environmental contexts specifically promoting cell-cycle arrest, apoptosis, glucose metabolism, and stress resistance [β-catenin expression [2O2 results in the activation of the small GTPase Ral that determines the phosphorylation and activation of JNK and JNK-mediated phosphorylation of FOXO1 [β kinases inhibits FOXO function inducing cytoplasmic translocation and protein degradation [The FOXO subfamily of forkhead transcription factors is emerging as a fundamental regulator of sistance –109. Kopsistance  and Essesistance  demonstrpression . FOXO acradation , 111–114β-catenin binds directly to FOXO through an evolutionary conserved mechanism and enhances FOXO transcriptional activity in mammalian cells [β-catenin, thereby inhibiting TCF transcriptional activity [β-catenin then appears to fulfill a critical function in balancing TCF signaling (mainly proliferative) and FOXO signaling (mainly devoted to stress response and apoptosis).an cells . In streactivity , 116. β-γ, β-catenin, and FOXO is demonstrated by several studies. The existence of an influence of Wnt/β-catenin on PPARγ expression has been reported in 2000 when Ross et al. [γ expression; however, the interaction between PPARγ and Wnt stretches out the adipogenesis and has been described in several processes where ROS production and oxidative stress are important such as aging, Alzheimer's and Parkinson's diseases, diabetes, hepatic stellate cells activation, liver fibrosis, and cancer [A cross-talk among PPARs et al.  described cancer , 118–121γ has been elucidated only years after the paper of Ross et al. [β-catenin induces the orphan nuclear receptor COUP-TFII [γ repressing its transcription [β [α [γ interacting with Wnt.The molecular pathway linking Wnt to the repression of PPARs et al. ; combinis et al.  demonstrOUP-TFII  that reccription . Other rcription , Smad/TFption [β , the Wntption [β , 120, anon [β [α , 137, maγ agonists such as TZD, GW1929, or 15d-PGJ2 inhibit β-catenin signaling in vitro and in vivo [γ activation induces cell-cycle arrest and apoptosis downregulating c-Myc through blockade of TCF activity [γ activation suppresses epithelial cell turnover and β-catenin transcriptional activity retaining the β-catenin in the cytosol [γ antagonist T0070907 promotes tumorigenesis in the small intestine and colon stimulating epithelial proliferation: this effect depends on increased expression of c-myc and Cyclin D1 genes and β-catenin activation [PPAR in vivo –126. In  cytosol . Converstivation , 138.γ directly interacts with β-catenin [β-catenin and PPARγ requires the presence of PPARγ co-receptor RXR [β-catenin and a catenin binding domain within PPARγ [γ may retain β-catenin in the cytosol so reducing its transcriptional activity [β-catenin expression after TZD treatment in vitro.Several studies have demonstrated that PPAR-catenin , 139. Thin PPARγ . It has activity  and may activity , 128; hoactivity  did not γ may indirectly increase β-catenin degradation influencing the phosphorylation status of GSK3β. PPARγ is a known activator of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphatase that inhibits AKT signaling [γ PTEN-mediated antagonism of AKT may reduce the inhibition of GSK3β enhancing proteasomal degradation of β-catenin, in agreement with other reports that have implicated PTEN as a negative regulator of β-catenin by way of AKT/GSK3β [Furthermore, PPARignaling . ConsequKT/GSK3β .γ-β-catenin-FOXO has been suggested by Almeida et al. during oxidative stress in the skeleton [γ expression increase with aging; the authors then go on demonstrating that ROS activate FOXO that decrease Wnt/β-catenin by competition; in turn, the ensuing PPARγ increased expression and activation further sink β-catenin signaling [γ-FOXO relationship.An axis PPARskeleton . The autignaling . Howeverignaling  did not γ at the mRNA and protein levels [γ promoter [γ activity through a transrepression mechanism that involves a direct protein-protein interaction [γ activation by its ligands and is mediated by a 31-amino-acid domain within FOXO1 that contains one inverted and two atypical LXXLL motifs [γ and RXR results in a dose-dependent inhibition of FOXO1-driven reporter activation [γ-FOXO; however it is not clear if the reported effect is due to a transrepression mechanism or is an indirect effect of PPARγ : RXR dimers.FOXO may modulate PPARn levels –131. In promoter , 131 anderaction , 131. Thtivation , creatinγ may regulate FOXO1 through modulation of AKT with a mechanism that may explain the signal cascade of insulin [γ, inducing PTEN, may block AKT activation and hence may, indirectly, increase FOXO activity. Interestingly, Sakamoto et al. [γ-PTEN-AKT that acts on FOXO with activating effects may also act on NRF2; however given that AKT increases NRF2 activity [γ-PTEN-AKT, conversely to the effect on FOXO, should reduce NRF2-induced transcription.It has been suggested that PPAR insulin ; this reo et al.  demonstractivity , the axiγ may influence FOXO acting on p38-MAPK, SIRT histone deacetylase, and the NF-κB. NF-κB and p38 inactivate FOXO and increase their ubiquitination-mediated degradation [γ may counteract the effects of both these pathways: in fact PPARγ transrepresses (and it is repressed by) NF-κB and influences the p38 MAPK signaling cascade increasing the expression of heat shock protein 27 (Hsp27) [κB [γ-dependent inhibition of FOXO1 [Moreover, PPARradation , 114. PP (Hsp27) . The Hsp (Hsp27)  and mighp27) [κB  and actip27) [κB , resp.).of FOXO1 .γ and subsequent repression on PPARγ transcriptional activity [γ by docking with its cofactors nuclear receptor co-repressor (NCoR) and SMRT [γ: Han et al. [γ and SIRT1 bind the SIRT1 promoter inhibiting the transcription of the deacetylase; moreover, PPARγ directly interacts with SIRT1 and inhibits SIRT1 activity; the association SIRT1-PPARγ appears to be mediated by the acetylation status of PPARγ [γ, it has been demonstrated that during senescence both acetylation and activity of PPARγ increase [γ acetylation might increase the activity. Interestingly, the increased acetylation of PPARγ may determine an inhibition of SIRT1 via direct binding, thus limiting the activating deacetylation of FOXO. Furthermore the inhibition of SIRT PPARγ-mediated may also explain the finding that PPARγ is involved in the senescence [Acetylation/deacetylation regulates different functions of FOXO proteins. It has been demonstrated that the deacetylase SIRT1 deacetylates FOXO proteins in response to stress , 148, inactivity . Furtherand SMRT . Finallyn et al.  demonstrof PPARγ . Acetylaincrease , 151 sugnescence . Paradoxnescence .A prolonged condition of oxidative stress, as a result of imbalance between antithetic prooxidation and anti-oxidation forces, may lead the cell to her apoptotic or necrotic doom.γ stands out as a central player.In this delicate and intricate game of equilibrium, when the cell may survive or die depending on her ability to sustain and repair the damage, PPARγ are produced during oxidative stress, PPARγ, if already expressed, may be one of the first responders directly inducing an arsenal of antioxidant molecules, inhibiting prooxidants and in the same time protecting the cells from apoptosis.Given that natural ligands of PPARγ does not act alone. Indeed the nuclear receptor is at the point of interconnection of various pathways, specifically the NRF2 and the Wnt/β-catenin and FOXO pathways (It is obvious that in this defense mechanism PPARpathways .γ and NRF2 are linked by a positive feedback loop that sustains the expression of both transcription factors and of antioxidant and prosurvival genes, as long as oxidative stress goes on. At the same time, the two genes exert a potent anti-inflammatory action inhibiting the NF-κB pathway.The redox-sensitive nuclear factor NRF2 plays vital role in cytoprotection against oxidative and electrophilic stress and is induced by oxidative stress. The pieces of evidence recollected in this paper demonstrate that PPARγ and the proproliferative Wnt/β-catenin pathway are associated by a negative feedback loop. The role of Wnt/β-catenin in response to oxidative stress is not straightforward: it has been demonstrated that the pathway is able to positively regulate the expression of some antioxidant genes and the absence of β-catenin increases the oxidative stress; however, in light of the interaction of Wnt/β-catenin with PPARγ, the inhibition of Wnt/β-catenin pathway may be a necessary step to block cell proliferation and to mount an antioxidant response inducing the expression of the anti-inflammatory and antioxidant PPARγ. Furthermore the block of Wnt/β-catenin pathway and the diversion of β-catenin from the cognate TCF towards the FOXO transcription factors increase not only oxidative stress response but also the rate of apoptosis.Conversely, PPARγ interacts also with FOXO. The two transcription factors elicit a positive response to oxidative stress but FOXO, binding with β-catenin, may induce apoptosis whereas PPARγ increases the expression of the antiapoptotic Bcl-2. The relation PPARγ-FOXO is ambivalent: FOXO directly inhibits PPARγ and indirectly induces it, sinking the negative effect of Wnt/β-catenin; on the other hand PPARγ acting exclusively indirectly is both inducer and repressor. This complicated relationship probably reflects the different effect on apoptosis, clearly proapoptotic FOXO, mostly antiapoptotic PPARγ. PPARγ may retard apoptosis inducing Bcl-2 and inhibiting FOXO; however this mechanism is self-limiting because activating FOXO, PPARγ creates a negative loop that may increase the chances of cellular death.PPARγ may be the bridge linking NRF2 to Wnt/β-catenin and FOXO pathways. Activating PPARγ, NRF2 not only potentiate the cellular oxidative response but may also overcome the negative feedback loop PPARγ-Wnt/β-catenin inducing cell cycle arrest giving more time to the cells to repair the damage; at the same time, through PPARγ, NRF2 may fine-tune the stress resistance and apoptotic FOXO-mediated responses to oxidative stress. However, because of the multiple regulatory links we have described, this interaction goes in both ways giving that Wnt/β-catenin and FOXO, inhibiting PPARγ, may influence NRF2 activity.Interestingly PPARγ in oxidative stress is essentially devoted to the quenching and containment of the damage and to foster cell survival; furthermore they depict a new signaling network NRF2-PPARγ-Wnt/β-catenin-FOXO that may be exploited for the treatment of oxidative-related diseases.Although the experimental pieces of evidence recollected in this paper have been obtained from various experimental models and conditions (see Tables γ promotes an antioxidant response integrating NRF2, Wnt/β-catenin, and FOXO pathways, but clearly the end result of oxidative stress, survival or apoptotic death, does not depend only on these sole genes but also on the net effect of other signaling pathways and regulatory circuits that converge on, and interact with, these transcription factors.In conclusion, during oxidative stress PPAR


Protein arginine methyltransferase-5 (PRMT5) is a chromatin-modifying enzyme capable of methylating histone and non-histone proteins, and is involved in a wide range of cellular processes that range from transcriptional regulation to organelle biosynthesis. As such, its overexpression has been linked to tumor suppressor gene silencing, enhanced tumor cell growth and survival.Quantitative real-time polymerase chain reaction, Western immunoblot and immunohistochemistry were used to characterize PRMT5 expression in lung cancer cell lines and human tumors. Clinicopathological findings of tissue microarray based samples from 229 patients with non-small cell lung carcinomas (NSCLC) and 133 cases with pulmonary neuroendocrine tumors (NET) were analyzed with regard to nuclear and cytoplasmic PRMT5 expression.There was statistically significant difference in PRMT5 messenger RNA expression between tumors and nonneoplastic lung tissues. Immunoblot experiments showed abundant expression of PRMT5 and its symmetric methylation mark H4R3 in lung carcinoma but not in non-neoplastic human pulmonary alveolar and bronchial epithelial cell lines. More than two thirds of lung tumors expressed PRMT5. High levels of cytoplasmic PRMT5 were detected in 20.5% of NSCLC and in 16.5% of NET; high levels of nuclear PRMT5 were detected in 38.0% of NSCLC and 24.0% of NET. Cytoplasmic PRMT5 was associated with high grade in both NSCLC and pulmonary NET while nuclear PRMT5 was more frequent in carcinoid tumors (p < 0.05).The observed findings support the role of PRMT5 in lung tumorigenesis and reflect its functional dichotomy in cellular compartments.http://www.diagnosticpathology.diagnomx.eu/vs/1611895162102528The virtual slides for this article can be found here: Lung cancer remains the greatest cause of cancer death, outnumbering the next three causes, colorectal, breast and prostate cancer combined. In 2011, it was estimated that more than 220,000 Americans would be diagnosed with lung cancer, and more than 150,000 people die from this disease. In partDNA methylation of tumor suppressor genes was identified as having equivalent functional consequences as mutation of key tumor suppressor genes,10. In cSimilar to DNMT, protein arginine methyltransferase 5 (PRMT5) has been shown to be involved in silencing of tumor suppressor genes promoting neoplastic transformation. PRMT5, along with PRMT7 and PRMT9, forms a group of type II PRMT enzymes that catalyze symmetric arginine methylation of histones and non-histone proteins,14. PRMTSince only limited data is available regarding the role PRMT5 in lung cancer, the goal of this study was to evaluate a large set of NSCLC and pulmonary neuroendocrine tumors (NET) for PRMT5 expression and the potential correlation of expression with clinicopathological variables.NSCLC cell lines  and small cell lung carcinoma cell lines (NCI-H69 and NCI-H719) were obtained from American Type Culture Collection , Table Six frozen tissue samples of adenocarcinoma (ADC) and 2 of squamous cell carcinoma (SQC) with matched nonneoplastic lung parenchyma (8 samples) were used for mRNA analysis. Three paraffin embedded samples of surgically resected lung ADC, 3 samples of SQC, 2 samples of small cell lung carcinoma (SCLC) and 1 large cell neuroendocrine carcinoma (LCNEC) were utilized for initial immunohistochemical analysis. The human tissues were collected in accordance with the institutional review board approved protocol.Possible correlations between PRMT5 expression and clinicopathological variables were analyzed utilizing tissue microarrays (TMA) representing NSCLC and pulmonary NET previously constructed from the archival material of Armed Forces Institute of Pathology,24. In sWestern immunoblot analysis was carried out according to standard protocol. Briefly, 30 μg of the total protein isolated from cells were used. Protein concentration was measured using a bicinchoninic acid protein assay kit . The primary antibody against PRMT5 (0.8 mg/ml) and Histone H4  were obtained from Abcam  and anti-β-actin from Santa Cruz Biotechnology . The secondary antibody was horseradish peroxidase–conjugated goat anti-rabbit or mouse IgG. Proteins were detected using enhanced chemiluminescence and films .Total RNA was isolated from frozen tissue using Trizol RNA isolation following the protocol supplied by the manufacturer . RNA samples were treated with DNase  to remove contaminating DNA and stored in -70°C freezer. Quantitation of mRNA expression was carried out using TaqMan real time polymerase chain reaction (PCR). Primers and probe used for the real time PCR analysis were obtained from Applied Biosystems . Reverse transcription was performed utilizing SuperScript II Reverse Transcriptase Kit . Briefly, 500 ng of template total RNA was reverse transcribed in a 15 μl reaction. The PCR amplification was conducted in 25 μl reaction using the TaqMan Universal PCR Master Mixture  according to the protocol supplied by the manufacturer. Real-time PCR was carried out in a 96-well plate using an Applied Biosystems 7900HT Sequence Detection System at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each sample was evaluated in triplicate and each reaction was repeated at least once to ensure reproducibility. The PCR cycle number at threshold (CT) was used for the comparison. The relative quantitative method was used for the quantitative analysis. Calibrator was the averaged ΔCt from the non-tumor tissues. Endogenous control was ribosome RNA 18S gene .IHC staining was performed in standard fashion on paraffin-embedded tissue. Paraffin embedded cell line pellet, resection and TMA block were cut at 4-microns and sections were placed on positively charged slides. Slides with sections were then placed in a 60°C oven for 1 hour, cooled, deparaffinized and rehydrated through xylenes and graded ethanol solutions to water. All slides were quenched for 5 minutes in a 3% hydrogen peroxide solution in water to block endogenous peroxidase. Slides then underwent heat-induced epitope retrieval employing Target Retrieval Solution  for 25 minutes at 96°C in a vegetable steamer (Black & Decker) and cooled for 15 minutes. Slides were then placed on a Dako Autostainer Immunostaining System. All incubations on the Autostainer were at room temperature. The primary rabbit polyclonal anti PRMT5 antibody  at 1:70 dilutions for tissues and at 1:150 for cell lines was incubated for 60 minutes. The two components of Mach 4 Universal Alkaline Phosphatase Polymer Kit  were applied sequentially for 15 minutes each. Staining was visualized with the Vulcan Fast Red chromogen . Slides were then counterstained in Richard Allen hematoxylin , dehydrated through graded ethanol solutions, cleared in xylene and coverslipped.Based on the expression patterns identified in the resection specimens, the tumor cell staining in TMA was evaluated in comparison to normal alveolar parenchyma. Since cytoplasmic expression was evenly distributed within a tumor but varied in intensity, the cytoplasmic expression was assessed as negative (lack of staining), low (weak staining) and high (strong staining). Since number of PRMT5 expressing nuclei varied within a tumor, the nuclear expression was assessed as negative (lack of staining), low (staining in less than 50% of nuclei) and high (staining in 50-100% of nuclei).t test was performed to compare the expression values in tumor samples with the values obtained from matched non-tumor samples. The associations between clinicopathologic variables and PRMT5 protein expression were examined with Pearson Chi-Square test. Survival probabilities were calculated using the Kaplan-Meier method and compared with the log-rank test. Prognostic significance of PRMT5 expression was evaluated with Cox regression model. Results with a p-value less than 0.05 were considered statistically significant. Data analysis was performed utilizing statistical package SYSTAT 13.0 .The mRNA expression levels were summarized graphically by plotting the mean and standard deviation of PRMT5 based on multiple amplifications. A Mean PRMT5 mRNA evaluated with Taqman real time PCR was 6.13 fold higher in NSCLC samples than that in matched non-neoplastic pulmonary parenchyma, p = 0.030 Figure a-c. In aPRMT5 expression was present in the majority of NSCLC subjected to immunohistochemical analysis Figure a-c. CytoPRMT5 expression was also seen in the majority of pulmonary NET Figure d-e. Cytoin vitro and in vivo studies suggesting importance of PRMT5 for proliferation of lung cancer cells.Epigenetic regulation plays an important role in oncogenesis, and histone modification has been recognized as one strategy for modifying epigenetic controls,26. It hin vitro observations that cytoplasmic function of PRMT5 relates to silencing of multiple growth promoting and cell death inducing molecular targets[Based on IHC analysis of more than 350 pulmonary tumors, our study also reveals that cytoplasmic PRMT5 is associated with higher grade in both NSCLC and pulmonary NET. Nuclear PRMT5 was more frequent in well-differentiated tumors (carcinoid tumors) than in poorly-differentiated tumors (SCLC and LCNEC). These findings further support the  targets,15. Simi targets. Based o targets. In ligh targets. The pre targets.Our study identified that within NSCLC, both cytoplasmic and nuclear PRMT5 expression was more frequent in squamous cell carcinomas than in adenocarcinomas, which is in line with prior observations that DNA methylation is universal phenomenon in lung squamous cell carcinomas,8. CytopIt has been recognized that genetic instability is common in pulmonary parenchyma adjacent to tumors. Shared genetic changes have been documented in tumors, precursor lesions and non-neoplastic lung parenchyma adjacent to tumors. ChromosIn summary, our study shows that PRMT5 is significantly overexpressed in neoplastic lung tissues supporting its role in lung tumorigenesis. Cellular localization of PRMT5 correlates with lung tumor grade/differentiation, supporting functional dichotomy of PRMT5 in cellular compartments.PRMT5: Protein arginine methyltransferase-5; HPAEpiC: Human pulmonary alveolar epithelial cells; HBEpiC: Human bronchial epithelial cells; TMA: Tissue microarray; NET: Neuroendocrine tumors; TC: Typical carcinoid; AC: Atypical carcinoid; LCNEC: Large cell neuroendocrine carcinoma; SCLC: Small cell lung carcinoma; ADC: Adenocarcinoma; SQC: Squamous cell carcinoma.The authors declare that they have no competing interests.KS, SS, RB, GAO: conceived and designed the study; XW, SS, WD: performed the experiments; KS, XW, MW, WD: analyzed the data; MV-C, JF, CLH, WZ: contributed reagents/materials/analytic tools; KS, MW, GAO: wrote/edited the paper. All authors read and approved the final manuscript.


We attained unusually high coverage of IKKβ, identifying an abundant site of Tyr phosphorylation at Tyr169 within the Activation Loop. The phosphomimic at this site confers a level of kinase activation and NFκB nuclear localization exceeding the iconic mutant S177E/S181E, demonstrating that RTK-mediated Tyr phosphorylation of IKKβ has the potential to directly regulate NFκB transcriptional activation. Signaling regulated by NFκB and related transcription factors is centrally important to many inflammatory and autoimmune diseases, cancer, and stress responses. The kinase that directly regulates the canonical NFκB transcriptional pathway, Inhibitor of κB kinase β (IKKβ), undergoes activation by Ser phosphorylation mediated by NIK or TAK1 in response to inflammatory signals. Using titanium dioxide-based phosphopeptide enrichment (TiO Receptor tyrosine kinases (RTKs) represent important signal transducers in the cell membrane and are comprised of nearly twenty families of homologous proteins in humans, with almost 60 distinct members . In the NFκB signaling, of central importance in human disease -11, is r2)-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS) . FGFR2  and IKKβAntibodies were obtained from the following sources: Bek/FGFR2 (C-17), IKKβ (H-4), IKKγ (FL-419), NFκB p65 (F-6), β-tubulin (H-235), GST (Z-5) from Santa Cruz Biotechnology; 4G10 (antiphosphotyrosine) from Upstate Biotechnology; Phospho-IKKα/β (Ser176/180) from Cell Signaling; horseradish peroxidase (HRP) anti-mouse, HRP anti-rabbit from GE Healthcare. The enhanced chemiluminence (ECL) reagents were from GE Healthcare. HEK293 cells were transfected and starved as described . Coimmun6 cells per 15cm tissue culture plate. 10 plates were transfected with FGFR2 and IKKβ expression plasmids as described -ATP supplemented with 20 μM ATP, and 0.5 μg of purified GST-IκBα(1-54) were incubated at 30°C for 30 min, and separated by 12.5% SDS-PAGE. Gels were stained, destained, dried and exposed to film. All kinase assays were repeated a minimum of 3 times, even those for which quantification is not presented. Where shown, quantification of a minimum of three replicate experiments is presented normalized against IKKβ S177E/S181E “EE” mutant, which was set at 100%. Error bars show standard error of the mean. HEK293 cells were transfected and starved overnight prior to lysing in Cytoplasmic Extract Buffer . Purified proteins were combined in Binding Buffer  and allowed to incubate with rotation overnight at 4°C. Amylose resin was added, samples incubated with rotation at 4°C for 2 hr then washed three times with Binding Buffer. Proteins were eluted with 10mM maltose in Binding Buffer, separated by 12.5% SDS-PAGE and transferred to Immobilon-P membranes for immunoblotting. Plasmids containing MBP-HLH+ and GST-FGFR2 fusion domains were transformed into Rosetta cells. Single colonies were grown to AMCF7 cells were transfected with Lipofectamine 2000 with no serum or antibiotics in media. Cells were collected 20 hr later for fractionation. Cell pellets were resuspended in CE Buffer . After vortexing and pelleting the cytoplasmic lysate was transferred to new tubes. Nuclear pellets were washed twice with CE Buffer before adding NE Buffer . Pellets were frozen on dry ice and thawed three times. Nuclear extracts were collected by centrifugation. Protein concentrations were determined by Bradford Assay. Equal amounts of total protein were separated by 15% SDS-PAGE and transferred to Immobilon-P membrane for immunoblotting.Figure S1Invitro binding of FGFR2 and IKKβ.Invitro binding assay determines the domain of FGFR2 which interacts with IKKβ. (A) A schematic of FGFR2 is shown. The extracellular domain contains three Ig-like regions (Ig) followed by the transmembrane domain (TM). The intracellular regions were isolated into domains: the juxtamembrane (JM), the 1st and 2nd kinase (KD1 and KD2) and the C terminal (Cterm) domains, each of which were fused in frame to glutathione-S-transferase (GST). The amino acids of FGFR2 that were used in the fusion proteins are indicated. (B) A schematic of IKKβ is shown with the N-terminal kinase domain (KD), the ubiqutin-like domain (ULD), the scaffold/dimerization domain (SDD) which also contains the leucine zipper (LZ) and helix-loop-helix (HLH) regions. The NEMO binding domain (NBD) is at the C-terminus of IKKβ. The region of IKKβ that was used in the MBP fusion protein, which includes HLH is indicated. (C) GST-FGFR2 and MBP-HLH+ fusion constructs were bacterially expressed and purified as described in Materials and Methods. The purified MBP-HLH+ protein was incubated with the GST-FGFR2 fusion proteins overnight and complexes were recovered with amylose resin. After washing, the proteins that bound to MBP-HLH+ were eluted with buffer containing maltose. Samples of the input GST-FGFR2 fusion proteins and the elutions were separated by 12.5% SDS-PAGE, transferred to Immobilon-P membrane and immunoblotted with anti-GST sera. Controls for the nonspecific binding of the GST-KD1+ and GST-KD2 proteins to the amylose resin were performed and only showed binding in the presence of MBP-HLH+ protein (data not shown). (PDF)Click here for additional data file.Figure S2Location of All Unique IKKβ Peptides and Phosphopeptides Identified. The IKKβ amino acid sequence is shown together with the location of all identified peptides and phosphopeptides with respect to structural motifs [l motifs . Boundar(PDF)Click here for additional data file.Table S1Description of Protein Samples Used for Mass Spectrometry.(PDF)Click here for additional data file.Table S2Threonine Phosphorylation of IKKβ: Analysis of Peptide Data.(PDF)Click here for additional data file.Table S3Serine Phosphorylation of IKKβ: Analysis of Peptide Data.(PDF)Click here for additional data file.Table S4Peptide Analysis of IKKβ: Total Peptides.(PDF)Click here for additional data file.Table S5Serine Phosphorylation Sites by Spectral Counts.(PDF)Click here for additional data file.


The use of biomarkers within the procedures of the Committee of Orphan Medicinal Products (COMP) of the European Medicines Agency (EMA) is discussed herein. The applications for Orphan Medicinal Product designation in the EU are evaluated at two stages. At the time of orphan designation application, the file undergoes an assessment to establish whether the proposed condition is a distinct and serious condition affecting not more than 5 in 10,000 people in the EU, and whether the product is plausible as a therapy for that condition. In cases where therapies already exist, the significant benefit of the candidate product over existing therapies is also evaluated. The orphan criteria are reassessed at the time of marketing authorisation, so that marketing exclusivity for the product in the orphan medical condition can be granted. Within this context, biomarkers have been used in submissions in order to define an orphan condition and to justify that the criteria for orphan designation are met. The current work discusses specific examples from the experience of the COMP, where biomarkers have played a decisive role. Importantly, it identifies the proposal of sub-sets of non-rare conditions based on biomarkers as a challenging issue in the evaluation of applications. In particular two specific requirements for the candidate orphan medicines in relation to the biomarker-based subsets are highlighted: the “plausible link to the condition” and the “exclusion of effects outside the subset”. The use of biomarkers, which are specific for medical conditions, is envisioned to accelerate the research and development and increase the success rate of pharmaceutical products . This apThe Committee of Orphan Medicinal Product (COMP) of the European Medicines Agency (EMA) has always faced challenges when evaluating submissions for orphan designation. The COMP has been assessing the use of biomarkers by sponsors of candidate orphan products in three main areas:•to define the distinct medical condition or a valid sub-set for the designation (orphan condition as described in guideline ENTR6283/Rev03) [•to justify the intention to diagnose, prevent or treat a condition with a product, (as per the provisions of Article 3(1)(a) of Regulation (EC) No 141/2000 [•to determine significant benefit (as per Article 3(1)(b) of Regulation (EC) No 141/2000 [In recent years, concomitantly with an increase in the number of applications , the conThis work was produced to present the current perspectives regarding the use of biomarkers in submissions for orphan designation. It is based on an internal EMA-COMP reflection paper produced by an ad-hoc biomarkers working group composed of Members of the COMP and its objectives include:•Examination of the role of biomarkers in submissions from sponsors seeking Orphan Medicinal Product Designation.•Presentation and discussion of past submissions to the COMP where biomarkers were used by sponsors in their request for an Orphan Medicinal Product Designation•Reflection on when and how the COMP has taken biomarkers into consideration when assessing medicinal products.Table The COMP follows certain guidance documents to assist it in evaluating submissions for Orphan Medicinal Product Designation. One of the principle guidance documents of this nature is the section on General requirements and Special considerations for the description of the condition in an application for orphan designation of Guideline ENTR 6283/00 Rev.03 [Typically only recognised distinct medical entities are considered as valid conditions for designation, while sub-sets are only exceptionally considered. Proposals of subsets based on biomarkers are scrutinized inter alia with regards to the following conditions:•the subset proposed should fall entirely within a distinct medical condition •there should be a clear delineation of the subset from the entire of the population (in the context of this paper: a well-defined biomarker)•the subset should have a “plausible link to the condition” and•the subset should be “closely linked to the pharmacological action of the medicinal product in such a way that the absence of these characteristics will render the product ineffective in the rest of the larger population with the same condition”In particular with regards to the requirement of “plausible link to the condition”, this is to be interpreted as a limitation to the category of subsets: the subset may not refer to features that do not pertain directly to the condition or disease, thereby limiting the never-ending potential sub-setting based e.g. on immunological or genomic features of the individual patients and not of the underlying disease.The requirement of “exclusion of effects outside the proposed subset” is equally important, and involves a vigorous assessment of the pharmacodynamics of the product to ensure that in the excluded population (in the context of this paper the population outside the biomarker-defined subset) the product will not have any favourable pharmacodynamic effect. To illustrate these limitations in the use of biomarker for the purpose of proposing subsets as valid conditions for designation, specific examples are presented below. These were selected on the basis of clarity and usefulness for the points discussed in this paper.There is a number of unsuccessful cases of orphan applications evaluated by the COMP, which involved biomarkers used to define specific orphan conditions. The justification of “a plausible link to the proposed orphan condition” and the “exclusion of effects” in the wider non-orphan population have proven to be the key drawbacks in these applications. Both of these points are illustrated in the actual case studies elaborated below. As only one of these products was not withdrawn prior to the final opinion from the COMP, the detailed aspects of confidential nature are not discussed in detail.The first example pertains to a case discussed in 2013, involving a therapy proposed for treatment of non-small-cell lung cancer in patients expressing HLA-A2.The Committee considered that in particular the “plausible link to the condition” Table  was not Two unsuccessful applications are discussed herein, one of which reached the point of formal adoption of a negative opinion that has been made public.a. In the first case discussed in 2012, a product was proposed for treatment of P-gp positive breast cancer. The sponsor was proposing that P-gp positive breast cancer could be considered a distinct medical condition under the orphan legislation as this breast cancer population is multidrug resistant due to clinically relevant high level of P-gp. The sponsor in the application further clarified that the targeted population covers only 25% of P-gp positive breast cancers, as this is the proportion they expected to be multidrug resistant. The COMP noted that P-gp may be found in close to 100% of breast cancer patients, and that the product could have an effect in a much larger population group than the specific subset proposed by the sponsor as being a distinct orphan medical condition. The COMP considered the 25% cut-off point to be arbitrary and unsupported by current clinical evidence to support the claim that this was a distinct orphan medical condition. The Committee concluded that the condition applied for (treatment of P-gp positive breast cancer) is not a valid subset for orphan designation.b. Another interesting case was discussed in 2011, pertaining to the application of a product for the treatment of ALK-positive non-small cell lung cancer. In that case the sponsor was proposing that the subset of “ALK-positive non small cell lung cancer” could be considered as a distinct orphan medical condition. This subset is characterised by a gene fusion between the anaplastic lymphoma kinase (ALK) gene and echinoderm microtubule-associated protein-like 4 (EML4), leading to expression of aberrant ALK receptors on the malignant cells. The proposed product for designation was claimed to be a specific inhibitor of ALK. However during the evaluation process it was noted that this product was also an inhibitor of the c-Met/HGFR receptor tyrosine kinase. The mixed nature of product actions established the basis that the proposed sub-setting was not justified since some effects may be anticipated in patients not bearing mutations or ALK overexpression.As per article 3 of Regulation (EC) No. 141/2000 the sponsor has to establish that the product is “intended for the diagnosis prevention or treatment” of a condition. This intention to diagnose, prevent or treat is examined under the broader term of “medical plausibility” in the proceedings of the COMP, and the relevant guidelines [significant benefit to those affected by the condition.In addition, significant benefit in Article 3(1)(b) of Regulation (EC) No. 141/2000 requires that in the case where a satisfactory method of diagnosis, prevention or treatment of the condition exists, the sponsor has to establish the medicinal product will be of Significant benefit (SB) is further defined in Article 3 of Commission regulation EC 847/2000 as “… a clinically relevant advantage or a major contribution to patient care” [In order to justify the rationale for the intention to prevent, treat or diagnose, and for the justification of a clinically relevant advantage in case other satisfactory methods exist, biomarkers have been used as surrogate endpoints, without major issues with their use having been identified so far. The sponsor may use them, as long as it justifies that the models or clinical settings are relevant for the proposed condition, and that the endpoints studied are appropriate. A long list of examples, can be found in the monthly release of the COMP plenary meeting minutes that are being made public by the European Medicines Agency since September 2012.In the context of orphan regulatory procedures, the single major challenge in biomarker use has been the sub-setting of broader medical entities in order to define a valid orphan condition for designation.With reference to the guideline on the format and content of the applications for designation, patients in a proposed sub-set have to present distinct and unique pathophysiological, histological, aetiological and clinical symptoms which are evaluable characteristic(s) with a plausible link to the proposed orphan condition and the biomarker characteristics should be essential for the medicinal product to carry out its action. Consequently two main requirements in the sub-setting of broader medical entities for the purpose of proposing orphan conditions emerge: 1) establishing a plausible link to the broader sub-setted condition and 2) demonstrating clearly that the presence or absence of the biomarker has a one-to-one relationship with the presence or absence of the drug to execute its pharmacodynamic activities.The “plausible link” requirement may be viewed as an argument against proposing subsets of non-rare conditions in the era of personalized medicine: when the biomarker refers not to the condition but to the individual patient this link is not preserved. At the same time, it may be viewed as a guardian of evolution of our scientific understanding: when the biomarkers refer to the condition then the link is confirmed, and this is of particular importance in cases when biomarkers redefine the classification of medical entities as our understanding evolves (diagnostic biomarkers). The specific nature of biomarkers may be associated with particular regulatory difficulties. For example, biomarkers used in the context of receptors on cancer cells, present a particular challenge as their detection capability may evolve by further development of newer detection assays. The tests are in time rendered more specific and sensitive and could help in identifying populations who will respond more effectively to the therapy but who do not necessarily represent a distinct patient population under the definition of the orphan legislation. Another example, indicative of the complexity is the tyrosine kinase area: the fact that an applicant for an orphan designation does not identify cross reactivity with other targets does not prevent that a wider investigation and testing of more cell lines or biopsies may eventually detect some cross reactivity. This would again challenge the volume of the subset compared to the underlying broader distinct medical entity. These examples illustrate the problem of unequivocally setting thresholds for definitions of acceptable sub-sets based on biomarkers.To take the argument a bit further, it is also becoming apparent that our scientific understanding underpinning the usefulness of biomarkers has been changing and evolving over time. Indicatively in neurofibromatosis with children it was presumed that tamoxifen should be used if the tumours were oestrogen receptor positive. But later it appeared that the product was not working only via the receptor and that also oestrogen receptor negative patients could benefit. A similar case seems to be with the KRAS mutation in colorectal cancer. Originally it was thought cetuximab would only be working in wild type KRAS patients but there are some other studies indicating that at least some KRAS mutation patients will benefit as well.Lastly, an interesting point is the evolution of the standards in the proceedings of the COMP, in the context of the increasing complexity of medicinal products reviewed and the recent advancements in scientific and medical research. Compared to the early days of the Committee, this has crystallised into shifting the focus from mainly the “exclusion of effects outside the subset”, to equally examining both the “exclusion of effects” and the “plausible link” principles with equal impact. This is clearly reflected in the examples described above.•Sub-setting of medical entities to define a valid orphan condition and justification of the criteria for orphan designation have been the main areas in which the COMP has used biomarkers and clinical surrogate endpoints.•If biomarkers are to be taken into account for the proposal of a sub-set of a broader distinct medical entity as a valid condition for designation, a decisive factor is the “plausible link to the condition”. The biomarker should not refer, in that case, to features external to the broader distinct medical entity.•In addition, if biomarkers are to be taken into account for sub-setting, the anticipated presence or absence of any pharmacodynamic effect, in either preclinical or clinical settings, is to be justified and supplemented by a convincing scientific argumentation and data justifying that the product will not work in the excluded patients.•Even though biomarkers can define a valid sub-set of a condition acceptable for designation, there is still a need to demonstrate medical plausibility and significant benefit in the defined condition.•As per the orphan regulation, at the time of marketing authorisation the criteria for orphan designation are reassessed, which also means that the validity of the used biomarker(s) has to be proven at that time.EMA: European Medicines Agency; COMP: Committee of Orphan Medicinal Products; HLA: Human leukocyte antigen; P-gp: P-glycoprotein; ALK: Anaplastic lymphoma kinase; KRAS: v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog.No competing interests are declared.All authors contributed equally to this work. All authors read and approved the final manuscript.


CTNND2), which encodes a scaffold protein in humans, has been found in a few malignancies. However, the expression pattern and contribution of δ-catenin to astrocytoma progression are unclear.δ-Catenin . In addition, CTNND2 overexpression promoted proliferation, invasion and Rac1 activity of U251 astrocytoma cells. Treatment of δ-catenin-transfected cells with a Rac1 inhibitor decreased Rac1 activity and invasion. δ-Catenin knockdown in U87 glioblastoma cell decreased cell proliferation, invasion and Rac1 activity.δ-Catenin protein expression was detected in cytoplasm of astrocytoma cells by immunohistochemistry. Analysis showed that grade I astrocytoma  and glial cells from normal brain tissue exhibited negative staining. δ-Catenin expression was significantly higher in grade III-IV  compared to grade II astrocytoma cells ; The results suggest that δ-catenin expression is associated with the malignant progression of astrocytoma and promotes astrocytoma cell invasion through upregulation of Rac1 activity. δ-Catenin expression levels may serve as a useful marker of the biological behavior of astrocytoma cells. Astrocytoma arises from neural stem or progenitor cells in the central nervous system. It is the most common primary brain tumor and accounts for approximately 60% of all brain tumors. Despite combined treatment strategies, which include surgery, radiotherapy and chemotherapy, the prognosis for high-grade astrocytoma, especially glioblastoma multiforme, has changed little over the past 10 years, with a median survival of only approximately 1 year ,3. Thus,The expression of δ-catenin was initially found to be limited to brain neurons and it can bind to presenilin 1, which is involved in the progression of Alzheimer's disease -6. δ-CatIn the present study, we examined the expression of δ-catenin in 156 astrocytoma tissue specimens and analyzed the correlation between its expression and clinicopathological factors. We also investigated the effect of δ-catenin on Rac1 activity and the biological behavior of astrocytoma cell lines.The study protocol was approved by the institutional review board of China Medical University. Primary tumor specimens and adjacent normal brain tissue were obtained from 156 patients diagnosed with astrocytoma who underwent resection in the First Affiliated Hospital of China Medical University between 2005 and 2008. None of the patients had received radiotherapy or chemotherapy before surgical resection. There were 95 male (60.9%) and 61 female (39.1%) patients with a median age of 43 years (range 15-67 years). The histological diagnosis and differentiation grade were evaluated for sections stained with hematoxylin and eosin according to the World Health Organization (WHO) classification guidelines. All 156 specimens were re-evaluated with respect to histological subtype and tumor grade. Tumors were graded and classified into grade I (11), grade II (61), grade III (51), and grade IV (33) according to WHO guidelines (2007).U251 and U87 cell lines were obtained from American Type Culture Collection . The cells were cultured in DMEM  containing 10% fetal calf serum (Invitrogen), 100 IU/ml penicillin , and 100 μg/ml streptomycin (Sigma). Cells were grown on sterilized culture dishes and were passaged every 2 days with 0.25% trypsin (Invitrogen). The pCMV5-FLAG/δ-catenin plasmid was a kind gift from Dr. Shun-ichi Nakamura at Kobe University and was transfected into cells using Attractene Transfection . A pCMV5 empty vector was used as a negative control. DharmaFECT1 reagent was used for siRNA transfection  according to the manufacturer's instructions. The protein level was assessed 48 h later by western blotting. The δ-catenin siRNA sequences were 5'-CUA CGU UGA CUU CUA CUC AUU-3' and 5'-UGA GUA GAA GUC AAC GUA GUU-3' and were transfected into U87 cell lines, which express relatively high levels of δ-catenin. Nonsilencing siRNA sequences were used as a negative control (5'-UUC UCC GAA CUU GUC ACA UUU-3' and 5'-AUG UGA CAA GUU CGG AGA AUU-3').Surgically excised tumor specimens were fixed with 10% neutral formalin and embedded in paraffin, and 4-μm-thick sections were prepared. Normal brain neurons present in the tumor slides was used as positive control. Immunostaining was performed using the avidin-biotin-peroxidase complex method . The sections were deparaffinized in xylene, rehydrated with graded alcohol, and then boiled in 0.01 M citrate buffer (pH 6.0) for 2 min in an autoclave. Hydrogen peroxide (0.3%) was applied to block endogenous peroxide activity and the sections were incubated with normal goat serum to reduce nonspecific binding. Tissue sections were incubated with δ-catenin mouse monoclonal antibody . Mouse immunoglobulin (at the same concentration as for the antigen-specific antibody) was used as a negative control. Staining for both antibodies was performed at room temperature for 2 h. Biotinylated goat antimouse serum IgG was used as a secondary antibody. After washing, the sections were incubated with streptavidin-biotin conjugated with horseradish peroxidase, and the peroxidase reaction was developed with 3,3'-diaminobenzidine tetrahydrochloride. Counterstaining with hematoxylin was performed and the sections were dehydrated in ethanol before mounting.Two independent blinded investigators examined all tumor slides randomly. Five views were examined per slide, and 100 cells were observed per view at 400 × magnification. Immunostaining of δ-catenin was scored on a semiquantitative scale by evaluating in representative tumor areas the intensity and percentage of cells showing significantly higher immunostaining than control cells in normal brain tissues. Cytoplasmic immunostaining in tumor cells was considered positive staining. We counted 400 tumor cells and calculated the percentage of positively stained cells. The proportion of cells exhibiting δ-catenin expression was categorized as follows: 0, absent; 1, 1-25%; 2, 26-50%; 3, 51-75%; and 4, ≥ 76%. The staining intensity was categorized as follows: 0, negative; 1, moderate; and 2, strong. The proportion and intensity scores were then multiplied to obtain a total score. To obtain final statistical results, a score < 2 was considered negative and scores ≥ 2 were considered positive.Total proteins from tissue and cells were extracted in lysis buffer  and quantified using the Bradford method. Samples of 50 μg of protein were separated by SDS-PAGE. Samples were transferred to polyvinylidene fluoride membranes  and incubated overnight at 4°C with antibody against δ-catenin  and a mouse monoclonal antibody against β-actin . After incubation with peroxidase-coupled antimouse IgG (Santa Cruz) at 37°C for 2 h, bound proteins were visualized using ECL (Pierce) and detected using a BioImaging System . Relative protein levels were quantified using β-actin as a loading control.U251 and U87 cells were transfected with siRNA or a δ-catenin plasmid for 48 h and then plated into three 6-cm cell culture dishes (1000 per dish) and incubated for 12 days. Plates were washed with PBS and stained with Giemsa. The number of colonies with more than 50 cells was counted. The colonies were manually counted using a microscope.Cells were plated in 96-well plates in medium containing 10% FBS at approximately 3000 cells per well 24 h after transfection. For quantitation of cell viability, cultures were stained after 4 days in MTT assays. In brief, 20 μl of 5 mg/ml MTT (thiazolyl blue) solution was added to each well and incubated for 4 h at 37°C. The medium was removed from each well and the resulting MTT formazan was solubilized in 150 μl of DMSO. Each solution was measured spectrophotometrically at 490 nm.5 cells and incubated for 16 h. Medium supplemented with 10% FBS was added to the lower chamber as the chemoattractant. Then non-invading cells on the upper membrane surface were removed with a cotton tip and cells that had passed through the filter were fixed with 4% paraformaldehyde and stained with hematoxylin. The number of invaded cells were counted in 10 randomly selected high-power fields under a microscope. The experiments were performed in triplicate.Cell invasion assays were performed in 24-well Transwell chambers with a pore size of 8 μm and the inserts were coated with 20 μl of Matrigel . At 48 h after transfection, cells were trypsinized and transferred to the upper Matrigel chamber in 100 μl of serum-free medium containing 3 × 10Rac1 activity was measured using a Active Rac1 Pull-Down and Detection Kit (Pierce). The Rac1 inhibitor NSC23766 was from Calbiochem . Cells were treated for 6 h at a concentration of 50 μM.2 test was used to examine possible correlations between δ-catenin expression and clinicopathologic factors. Student's t-test was used to compare densitometry data on focus numbers between control and δ-catenin-transfected cells. All p values are based on a two-sided statistical analysis, and p < 0.05 was considered to indicate statistical significance.SPSS version 11.5 for Windows was used for all statistical analyses. A χIn cells from normal brain tissue, only neurons showed strong cytoplasmic (include axon) expression of δ-catenin, whereas glial cells showed no expression Figure . Cytoplap < 0.05).We examined the difference between the expression pattern of δ-catenin according to tumor location. As shown in Table To determine whether δ-catenin enhances the proliferation and invasion of astrocytoma cells, we carried out δ-catenin transfection in the U251 astrocytoma cell line. siRNA knockdown was performed in the U87 glioblastoma cell line.p < 0.05; Figure p < 0.05; Figure p < 0.05; Figure p < 0.05). δ-Catenin knockdown also decreased Rac1 activity .To explore whether the effect of δ-catenin on cell invasion was dependent on Rac1 activity, we measured cell invasion and Rac1 activity after treatment of δ-catenin-transfected cells with the Rac1 inhibitor NSC23766. The inhibitor abolished the effect of δ-catenin on cell invasion (δ-catenin 73 ± 15 vs δ-catenin + Rac1 inhibitor 42 ± 9, δ-catenin plays an important role in dendritic morphogenesis, which is associated with changes in the activity of small GTPases ,13. The Analysis of mRNA in primary cultures of neurons and glial cells prepared from fetal rat brain showed that δ-catenin expression levels were higher in neurons than in glial cells . HoweverTo explore the relationship between δ-catenin and the biological behavior of astrocytoma cells, we transfected a δ-catenin plasmid into the U251 cell line, which has low δ-catenin expression, and knocked down δ-catenin in the U87MG cell line, which has high δ-catenin expression. We found that δ-catenin overexpression caused a significant increase in the proliferation rate, colony formation and invasion ability of U251 cell lines, with a significant increase in Rac1 activity, which was in accordance with our previous results for lung cancer . On the In conclusion, overexpression of δ-catenin in astrocytoma cells was correlated with higher grade and δ-catenin was identified as an important oncoprotein in maintaining the malignant behavior of glioma cells. Overexpression of δ-catenin activated Rac1 and promoted astrocytoma cell proliferation and invasion. Thus, δ-catenin is a candidate target protein for future astrocytoma therapeutics.The authors declare that they have no competing interests.WM and ZD carried out the immunohistochemistry, cell biological analysis and immunoassay. DQ participated in the design of the study and drafted the manuscript. WY participated in its design. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/514/prepub


Chemotherapy for lung cancer can have a detrimental effect on white blood cell (WBC) and red blood cell (RBC) counts. Physical exercise may have a role in improving WBCs and RBCs, although few studies have examined cancer patients receiving adjuvant therapies. The purpose of this pilot trial was to examine the effects of an exercise intervention utilizing resistance bands on WBCs and RBCs in lung cancer patients receiving curative intent chemotherapy.A sample of lung cancer patients scheduled for curative intent chemotherapy was randomly assigned to the exercise intervention (EX) condition or usual care (UC) condition. The EX condition participated in a three times weekly exercise program using resistance bands for the duration of chemotherapy.p = .008; d = 1.68). There were no significant differences in change scores in RBCs.A total of 14 lung cancer patients completed the trial. EX condition participants completed 79% of planned exercise sessions. The EX condition was able to maintain WBCs over the course of the intervention compared to declines in the UC condition (Exercise with resistance bands may help attenuate declines in WBCs in lung cancer patients receiving curative intent chemotherapy. Larger trials are warranted to validate these findings. Ultimately these findings could be informative for the development of supportive care strategies for lung cancer patients receiving chemotherapy.NCT01130714.Clinical Trials Registration #:  Chemotherapy for lung cancer can have a detrimental effect on both red blood cell (RBC) and white blood cell (WBC) counts due to bone marrow suppression. Declines in WBC subsets can result in delays in chemotherapy and reductions in dosage  and randomly assigned with a computer-generated program to either the EX or UC conditions in a 1:1 ratio in blocks of 4. An outside research assistant created the allocation sequence and assembled the results into a series of sequentially numbered opaque envelopes. The results were concealed from the research team until the next consecutive envelope was opened by a research assistant in the presence of the study participant.The study was approved by the University and Medical Center Institutional Review Board at East Carolina University. Participants were recruited from a cancer center in Eastern North Carolina and gave written and informed consent in English before commencing the trial . Eligibility criteria included (a) histologically confirmed diagnosis of non-small cell or small cell lung cancer, (b) eligible for chemotherapy with curative intent, (c) 21 years of age or older, and (d) approved to participate in trial by treating oncologist. Individuals were excluded if they had already commenced chemotherapy to treat lung cancer. All baseline and post intervention assessments were completed at the cancer center.Participants randomized to the EX condition participated in the exercise program for the duration of their chemotherapy starting one week after the first cycle of chemotherapy began until 3 weeks after the last cycle . The exercise program consisted of an exercise routine with resistance bands that consisted of total body exercises three times per week with at least one day of rest between sessions. Exercises simulated traditional resistance exercises with free weights and included biceps curls, shoulder pull backs, seated rows, triceps extensions, chest press, leg extensions, hip flexion, hip abduction, hip adduction, heel raises, and chair squats. Resistance bands are portable, inexpensive, can be used for a variety of total body exercises, and have been used successfully with older populations  were assessed at baseline (within one week of commencing the first cycle of chemotherapy) and post intervention (three to four weeks following the last delivery of chemotherapy). EX condition participants were instructed to not complete any exercise sessions within 24 hours of their scheduled post intervention blood draw.White and red blood cell counts were determined by routine CBC at the cancer center laboratory. Results were obtained from patient charts.Demographic and medical information were assessed from patient records  and questionnaire .Exercise level at baseline was determined by the Leisure Score Index (LSI) of the Godin Leisure-Time Exercise Questionnaire GLTEQ;  at α = .05. Comparisons between baseline demographic and medical characteristics and baseline outcome measures were examined by independent samples t-tests for continuous data and chi-square analyses for categorical data. Independent samples t-tests were used to calculate differences between the EX and UC conditions for WBC and RBC at post-intervention. Differences in change scores between groups were examined using independent samples t-tests. Effect sizes (d) were calculated by dividing the difference of the means by the pooled standard deviation . There were no significant differences in demographic and medical characteristics between the groups at baseline  and no significant differences in RBC . Based on the analysis of differences in change scores, WBC declined significantly less in the EX condition compared to the UC condition over the course of the intervention (p = .008) indicating a large effect size (d = 1.68). There were no significant differences in change scores in RBC between the EX and UC conditions and the effect size was small .Baseline, post intervention and change scores for RBCs and WBCs are displayed in Table There were three adverse events in the EX condition and one in the UC condition. In the EX condition one participant suffered a stroke (completed eight exercise sessions prior to the stroke), one was hospitalized for most of the intervention  and one withdrew due to increasing weakness and weight loss (completed eight exercise sessions before withdrawing). In the UC condition one participant suffered a myocardial infarction during the course of chemotherapy and was withdrawn from analyses.This pilot trial provides novel information about the effect of an exercise program utilizing resistance bands on blood counts in lung cancer survivors receiving curative intent chemotherapy. In partial support of our hypotheses, we found a significant difference in changes in WBC in favor of the EX condition. There were however no significant differences in changes in RBC between the EX and UC conditions.The positive effect of exercise training on WBC in this pilot trial is in line with past research that suggested that participation in an exercise program may improve WBC and subsets in cancer survivors during adjuvant therapy , the use of a weekly supervised exercise session as part of the protocol, and the relatively high adherence rate to the program (79%) by the EX condition. The limitations include the small, heterogeneous sample, differential exclusion from analyses in the EX condition, and missing data on some baseline variables for some participants.In summary, this pilot trial suggests that exercise training utilizing resistance bands may attenuate declines in WBC but not RBC counts in lung cancer survivors receiving curative intent chemotherapy. Moreover, the exercise program utilized may be safe and feasible for this population. Cancer care providers may be advised to consider chair based exercise programs with resistance bands as a potential supportive care strategy for lung cancer survivors. Future large scale trials are needed to further validate these results in both lung cancer and other cancer populations.