Hydra through to humans, and is expressed in diverse organisms, tissues and human disease contexts. Frizzled receptors can homo- or hetero-polymerise and associate with several co-receptors to transmit Wnt signalling. Notably, Frizzled7 can transmit signalling via multiple Wnt transduction pathways and bind to several different Wnt ligands, Frizzled receptors and co-receptors. These promiscuous binding and functional properties are thought to underlie the pivotal role Frizzled7 plays in embryonic developmental and stem cell function. Recent studies have identified that Frizzled7 is upregulated in diverse human cancers, and promotes proliferation, progression and invasion, and orchestrates cellular transitions that underscore cancer metastasis. Importantly, Frizzled7 is able to regulate Wnt signalling activity even in cancer cells which have mutations to down-stream signal transducers. In this review we discuss the various aspects of Frizzled7 signalling and function, and the implications these have for therapeutic targeting of Frizzled7 in cancer.Frizzled7 is arguably the most studied member of the Frizzled family, which are the cognate Wnt receptors. Frizzled7 is highly conserved through evolution, from  Drosophila have four, whilst C. elegans have three. Sequence analysis indicates that vertebrate Fzd genes can be divided into discrete classes based on structural homology. Fzd1, Fzd2 and Fzd7 share approximately 97% identity, Fzd5 and Fzd8 share 70% identity, Fzd4, Fzd9 and Fzd10 share 65% identity and Fzd3 and Fzd6 share 50% amino acid identity [Frizzled7 (FZD7) is located on human chromosome 2q33 and contains 3859 nucleotides that produce a 574 amino acid transmembrane protein, which belongs to a family of Wnt receptors. The Wnt signalling pathway regulates a diverse repertoire of cellular functions, playing critical roles during embryonic development, stem cell function and is often deregulated in cancer. Frizzled (Fzd) receptors have an extracellular N-terminal with a conserved cysteine rich domain (CRD), which is responsible for binding to Wnt ligands, seven transmembrane domains and an intracellular C-terminal with a putative PDZ binding domain. The number of Fzd genes varies between class of organism; mammals have ten Fzd’s, Drosophila and Xenopus demonstrate that Wnt1 is an important regulator of many cell functions during development including proliferation, migration, differentiation, apoptosis and cell polarity. These experiments, and subsequent ones, identified that Wnt1 is a homolog of Drosophila Wingless (Wg) and is the ligand for a signalling pathway which signals through Glycogen synthase kinase-3 beta (GSK3β), β-catenin and the transcription factors T-cell factor-4 (Tcf4)/Lymphoid enhancer binding factor-1 (Lef1), and was consequently called the Wnt pathway  [The Wnt1 gene  was first identified during a screen to identify oncogenes capable of inducing breast tumours in mice when mutated by infection with mouse mammary tumour virus (MMTV) . Since t “Int1”) . In addi “Int1”) .Drosophila suggest Wg can act as both a short and long-range inducer, which presents an interesting paradox; how do Wg ligands, which are hydrophobic and prone to form aggregates in an aqueous extracellular environment, act as long-range inducers if their lipid moieties confer high affinity for membrane tethering? Recent research from Roel Nusse’s lab has addressed this long standing question. Secreted Wg-interacting molecule (Swim), a member of the Lipocalin family of transporter proteins, can bind with nanomolar affinity to Wg, chaperoning Wg through the extracellular milieu, whilst preserving Wg solubility and signalling activity [Swim knockdown disrupts the identity of cells positioned distally from the local Wg source, demonstrating that they require long-range Wg signaling. As anticipated, Swim reduction resembles Wg mutant long-range phenotypes [Drosophila neuromuscular junction (NMJ) that Wg signal transmission can occur via exosome release, which contain the Wnt binding protein Evenness Interrupted (Evi) [Evi mutants phenocopy Wg mutant tissues [Wnt proteins are ~40 KDa in size and mediate relatively close-range signalling between cells. Recent evidence for the mechanism of this short-range signalling suggests that lipid modifications to Wnt by the palmitoyltransferase enzyme Porcupine in the endoplasmic reticulum target it for tethering to the membrane by the carrier protein Wntless . Thus exactivity . As suchenotypes . Furthered (Evi) . Evi is  tissues . Additio tissues . Besides tissues .Xenopus axis duplication for canonical signalling and orientation of hairs on the wing surface of Drosophila for non-canonical. The interaction between the Wnt ligand and the receptors it associates with on neighbouring cells determines which of these pathways is activated (2+ activation (discussed in detail below), Wnt5a in co-operation with Ror2 can inhibit Wnt3a-mediated canonical Wnt signalling through promoting β-catenin degradation independent of Gsk-3 [2+ signalling triggers calcium calmodulin dependent protein kinase II (CaMKII) activation, which induces the Transforming growth factor-beta-activated kinase 1 (TAK1)-Nemo-like kinase (NLK)-Mitogen-activated-protein-kinase (MAPK) pathway, shown to phosphorylate TCF/LEF factors and inhibit the interaction of the β-catenin/TCF complex with DNA. Thus, the TAK1-NLK-MAPK-like pathway, via Wnt5a/Ca2+ signaling, negatively regulates the Wnt/β-catenin signalling pathway [Wnt signalling is broadly divided into canonical and non-canonical pathways based primarily on whether signalling activates β-catenin , and associated phenotypes including transformation of mammary epithelial cells and ctivated . Some Wnof Gsk-3 ,16. Demo pathway ,18. Inte pathway .The pivotal effector of the canonical Wnt pathway is the co-transcription factor β-catenin. In the absence of active Wnt signalling, β-catenin is primarily localised to cell-cell adherens junctions and any free cytoplasmic β-catenin is rapidly phosphorylated by GSK3 in a degradation complex which targets β-catenin for proteosomal degradation. The destruction complex also includes Adenamatous polyposis coli (Apc), Casein kinase 1 (CK1) and Axin to facilitate phosphorylation of β-catenin. During canonical Wnt signaling, Wnt ligands bind to transmembrane Fzd receptors and low-density lipoprotein receptor-related protein 5 or 6 (Lrp5/6) co-receptors. Once activated, this receptor complex associates with the cytosolic scaffold protein Dishevelled (Dvl) to inhibit GSK3 enzyme activity. Consequently, β-catenin escapes phosphorylation and subsequent degradation, accumulates in the cytoplasm and translocates into the nucleus. β-catenin cannot bind to DNA directly but instead regulates transcription by associating with the transcription factor complex Tcf4/Lef1 that bind to conserved E-box sequences in regulatory elements of genes. Wnt1 and Wnt3a are prototypical examples of ligands that consistently activate this pathway .2+). PCP signalling is activated when Wnt ligands including Wnt5a [The non-canonical Wnt pathway is defined as being able to signal independent of β-catenin and has two arms; the Planar Cell Polarity (PCP) pathway and the Calcium pathway  is comprised of five secreted glycoproteins , which bind directly to Wnts, thereby altering their ability to bind to the Wnt receptor complex . All sFRThe crystal structures of the CRDs from mouse Fzd8 and sFRP3 reveal the potential to form dimers , which iIt should be noted that sFRPs do not always inhibit Wnt signalling, as nanomolar concentrations of sFRP1 increase Wnt3a signalling in HEK293 cells, whilst higher concentrations inhibit Wnt signalling . In addiSimilar to SFRPs, Wnt inhibitory factor 1 (WIF-1) is also able to bind to Wnt ligands and inhibit Wnt signalling . WIF-1 iWIF-1 inhibits Wnt signalling during development to help regulate Wnt expression in the paraxial mesoderm , and itsThere are four Dickkopf (Dkk) proteins in the vertebrate genome, Dkk-1, -2, -3 and -4. Dkk proteins selectively antagonise canonical Wnt signalling by binding to Lrp5/6, thus preventing Fzd and Wnt to form a ternary complex . They caR-Spondin (R-Spo) proteins are potent agonists of Wnt signalling, but only in the presence of Wnt ligands . There aThe highly related transmembrane E3 ubiquitin ligases, ZNRF3 and RNF43, act as negative regulators of canonical Wnt signalling that are integral to R-Spo/Lgr Wnt potentiation ,58. BothIn addition to its important role in regulating canonical Wnt signalling, R-Spo3, is able to regulate the Wnt/PCP pathway . R-Spo3 Apc is mutated in 80%–90% of colorectal tumours [Apc gene led them to propose the “just right” or Goldilocks model of Wnt signalling in cancer which proposes an optimal, but not excessive, level of Wnt pathway activation to transform cells, suggestive of constrained Wnt activation [Min/+Apc mice , and thus leaves open the possibility that Wnt signalling can be modified in cells with Apc mutations. Indeed, several publications in the mid 2000’s demonstrated that manipulation of factors up-stream of the degradation complex, at the level of the Wnt ligand and receptor, could alter the response of colorectal cancer cells even if they contained mutations to APC. For example, the promoter regions of SFRPs [APC mutant CRC tumour cells in a mouse xenograft model [The Goldilocks model also requires that the degradation complex is still partially functional even in cells with mutations in of SFRPs ,80,81 anof SFRPs  are hypeof SFRPs ,83. The ft model . Furtherft model . Importaft model ,86,87,88Drosophila, which was monitored by changes to the orientation of hairs on the wings surface. As such mutations in Fzd genes resulted in changes to the PCP pathway, which disrupted the orientation of wing hairs into a whorl-like appearance [The Fzd genes were originally discovered along with several other genes including dishevelled (dsh), fuzzy (fy) and multiple wing hairs (mwh), that affect planar cell polarity in pearance ,90. Sincpearance  and hydrpearance  to vertepearance , and havpearance ,94. Humapearance , and waspearance .Drosophila, intense research into the identification, expression and function of Wnt and Fzd homologues during development in other model organisms such as Xenopus, C. elgans and vertebrates (mice) ensued. Indeed, the successful cloning of Xenopus Fzd7 (Xfz7) by Wheeler and Hoppler [β-catenin [Fzd7, Gsk-3β, Dvl, Apc, and Axin from the Wnt/β-catenin pathway [Van Gogh, Flamingo and JNK from the Wnt/PCP pathway [Drosophila Fzd2) [Following the discovery of Fzd genes in  Hoppler  facilita Hoppler , gastrul Hoppler  and inte Hoppler . This is-catenin , Fzd7, G pathway ,100 and  pathway  along an pathway . The org pathway ,104,105.la Fzd2) ,106, whila Fzd2) .Xfz7 has been shown to regulate the coordinated convergent extension movement of cells [Xenopus laevis. Furthermore, the absolute requirement for canonical Wnt signalling in the developing and adult intestinal epithelium implicates a pivotal role for Fzd receptors [Indeed, of cells , tissue of cells  and dorsof cells  during geceptors ,108,109.eceptors ,110. As eceptors .−/−Fzd7 mice are viable and have a mild truncation and kink in the tail [−/−Fzd7 mice do not have an intestinal phenotype, this suggests that PCP signalling via Fzd7 is less important during intestinal development and homeostasis. Furthermore, canonical Wnt signalling is absolutely required during intestinal development and homeostasis [−/−Fzd7 mice during development and homeostasis of the intestine. Fzd2 has been demonstrated to compensate for loss of Fzd7 during development, since compound −/−Fzd2; −/−Fzd7 mice die at E10.5, whilst −/−Fzd1; −/−Fzd7 are healthy and fertile [Fzd7 can be tolerated during development and homeostasis of the intestine, surprisingly, acute genetic deletion of Fzd7 is deleterious in intestinal stem cells [the tail , which ithe tail ,114,115,eostasis . Therefo fertile . Althougem cells . This co−/−Fzd7 mice, the compensation mechanism using Fzd2 in the absence of Fzd7, has been occurring all through development and thus the system is fully established in the adult intestinal epithelium. This is a very different scenario from acute, conditional deletion which would result in significant stress for intestinal stem cells, which require Wnt signalling, possibly resulting in a different response from that of continued absence of Fzd7 from the beginning of conception as in a −/−Fzd7 mouse.Firstly, in Fzd7 in the intestinal epithelium triggers rapid repopulation of the epithelium with Fzd7 proficient cells, which is not possible in a −/−Fzd7 mouse [c-Myc [Chk1 [Fzd7 [−/−Fzd7 mice, however regeneration does recover and is associated with an increased expression of Fzd2 and Fzd1, suggesting that if repopulation with Fzd7 proficient cells is not possible (as in a −/−Fzd7 mouse), then compensation with other Fzd genes can occur [Secondly, conditional deletion of /− mouse . Conditie [c-Myc  and Chk1yc [Chk1 , also trk1 [Fzd7 , rather an occur .2+ pathways [Fzd7 is able to modulate the transmission of both canonical and non-canonical Wnt pathways. There are many examples of Fzd7 transmitting canonical Wnt signalling in several tissues during development, homeostasis and disease in many organisms including humans . This ocpathways .Drosophila wing imaginal disc epithelium. This study revealed that the intracellular C-terminal of Fzd proteins is responsible for directing the localisation of specific Fzd receptors within an apical-basal axis, which in turn direct either PCP  or canonical  Wnt signalling [+ stem cells [+ intestinal stem cells [in vivo [Another layer of complexity that can influence the activation of specific Wnt/Fzd pathways is the subcellular localisation of Fzd receptors. This was formally demonstrated in experiments overexpressing chimeric DFzd1 and DFzd2 proteins in the gnalling . Of noteem cells . This fiem cells ,122 on tem cells , and is [in vivo . It willXenopus development Fzd7 can form a complex with R-Spo3 to transmit Wnt5a signals and activate PCP signalling by inducing Sdc4-dependent, clathrin-mediated endocytosis [During ocytosis . These dXenopus development [Xenopus development [Xenopus gastrulation, suggesting it can regulate the Ca2+ pathway [Fzd7 can also co-operate with Ryk co-receptors to transmit Wnt 11-mediated signals via β-arrestin2-dependant endocytosis and JNK in convergent extension during elopment . Furtherelopment . Fzd7 ha pathway . Recentl pathway .Finally, the specification and expansion of human embryonic stem cells (hESCs) during development relies on controlled spatial and temporal Wnt signalling. Meta-analysis of 38 publications studying gene signatures of hESCs identified that FZD7 is consistently expressed highlighting that FZD7 is the predominant FZ receptor in many stem cell populations . This waXenopus Wnt8 bound to Fzd8 CRD revealed two distinct residue moieties that facilitate Wnt/Fzd binding [The Wnt-Fzd interaction is promiscuous, with a single Wnt able to bind multiple Fzd proteins, and vice versa, to activate canonical and/or non-canonical Wnt signalling. Successful crystallisation of  binding . One is  binding . The aut binding .2+ pathway [The Wnt-Fzd structure reveals the precision with which Wnt binds Fzd and provides evidence for complex oligomerisation with co-receptors to transduce signals to cytoplasmic signal transducers, most notably, Dvl . The Fzd pathway .2+ pathway in melanoma and is associated with a more aggressive phenotype [This promiscuity of Fzd7 to associate with different co-receptors and regulate both arms of the Wnt pathway may explain its frequent up-regulation in many different cancers to drive tumour growth, as both pathways are implicated in cancer. Although a variety of proteins have been discovered to bind Fzd7, not all of them have been functionally characterised  . Wnt/β-catenin signaling is one of the key signalling pathways shown to regulate FZD7 expression, as FZD7 expression is increased in human embryonic carcinoma cells following exposure to Wnt3a conditioned medium [FZD7 promoter houses putative TCF binding sites, confirming its regulation by Wnt/β-catenin signaling [FZD7 is further supported by findings demonstrating the activation and repression of a FZD7 promoter-driven luciferase reporter in response to β-catenin stimulation and inhibition respectively [Fzd7 expression is regulated by more than just Wnt signalling. For instance, unpublished data from our own lab indicates that Fzd7 is a transcriptional target of Jak/Stat3 signalling [FZD7 expression following pharmacological inhibition (AG490) of JAK2 [NOTCH3, was shown to regulate the expression of FZD7 in human mammary epithelial cells [FZD7 to be expressed predominantly by luminal progenitor cells, which is hypothesised to regulate luminal cell differentiation through a NOTCH3-FZD7 signalling axis [Gene regulation is governed by a complex network of cellular mechanisms, which can be modulated at every step. As such, the ever-expanding interplay between signaling pathways, transcription factors and post-transcriptional modifiers demonstrates that the expression of a single gene is not exclusively governed by a single factor. Indeed several recent studies have demonstrated the expression of d medium . In addiignaling . Wnt/β-cectively . Howevergnalling . These f of JAK2 . More real cells . Furthering axis .Fzd7 expression. For instance, miRNA-206 is a critical myogenic regulation factor required for correct skeletal muscle development and differentiation [Fzd7 to be a direct target of miRNA-206 [Fzd7 induces increased proliferation and abnormal myoblast differentiation [Furthermore, microRNAs (miRNAs or miRs), small non-coding RNAs that influence post-transcriptional gene expression through imperfect binding to the 3’ UTR of mRNA, play important roles in development and disease, placing them as potential therapeutic targets. As such, several miRNAs have been identified to influence the activation of Wnt signalling via regulation of ntiation . SubsequiRNA-206 . As suchntiation . These fntiation .Fzd7 [Fzd7 [−/−Fzd7 mice [Wnt signalling is a critical signaling pathway during the development, homeostasis and regeneration of the intestinal epithelium ,62,138. Fzd7 . Indeed,d7 [Fzd7 . These dFzd7 in several different cancers, and associated with reduced tumour growth, which are discussed in the relevant cancer sections below. However, recently Li et al. uncovered a two-faceted role for miRNA-126 in acute myeloid leukemia (AML), demonstrating that either overexpression or knockout of miRNA-126 could promote AML via inducing the expression of oncogenic fusion genes [ERRFI1 and SPRED1, but increased Wnt pathway activation via increased FZD7 expression. This resulted in accelerated AML progession, however, cells were sensitised to standard chemotherapy agents [Fzd7 expression, and illustrate its importance in several biological contexts, from development and regeneration through to homeostasis and cancer.Likewise, miRNAs have been shown to influence the expression of on genes . Interesy agents . CollectAPC were first observed in colon tumours of FAP  patients in the 1990’s [Apc caused multiple intestinal adenoma’s  [Apc throughout the intestine using transgenic mice resulted in rapid uncontrolled proliferation similar to tumourigenesis [Lgr5 were identified as the cell of origin for intestinal cancer, as truncation of Apc specifically in Lgr5+ cells resulted in widespread, rapid, development of adenoma’s throughout the small intestine and colon [β-catenin mutations are observed at much lower frequencies than Apc in intestinal tumours (~5% compared to ~75%) [Apc or oncogenic mutation in β-catenin are mutually exclusive but appear to be obligatory initiating steps for CRC [β-catenin are more common in intestinal tumours with microsatellite instability [APC truncation may provide the genetic make-up for additional regulation by the Wnt pathway  ,145, whiigenesis ,147. Furnd colon . Althougto ~75%) ,150, sup for CRC . Thus, i for CRC ,152. Onciewed in  and papein vitro , or usin[in vivo .Due to the high frequency of Apc mutations observed in intestinal cancer it was assumed that the Wnt receptor complex would not be important in regulating Wnt activity in this setting. However, as described above, SFRPs, WIF-1 and DKK1 are often hypermethylated and downregulated very early in intestinal cancer suggesting the Wnt receptor complex does play a role ,80,155. in vitro and in xenograft experiments with stably transfected SK-CO-1 cells, which harbour mutations to APC [Fzd7 in intestinal organoids resulted in crypt atrophy and organoid death, whilst conditional deletion of Fzd7 specifically in the Lgr5+ intestinal stem cells triggered rapid epithelial repopulation in transgenic mice [Several FZD receptors are over expressed in human intestinal tumours and cell lines, including FZD3, FZD6 and FZD7 ,106,158.s to APC . Recentls to APC . Conditinic mice , demonstin vitro and also completely block the formation of secondary liver tumours in mice following intrasplenic injection of SW620 cells transduced with the corresponding shRNA [In addition to its role in the very first stages of tumourigenesis, Wnt signalling is also implicated in the progression and invasion of intestinal tumours. Analysis of gene expression in the metastatic potential of human primary colon cancers identified several Wnt target genes to be important, including BAMBI, BOP1, CKS2 and NFIL3. Inhibition of these Wnt target genes with shRNA was able to reduce migration and invasion ng shRNA .Mph cells (human colon cancer cells) which go through cycles of EMT and MET in vitro [Mph cells, however FZD7 shRNA could also inhibit migration of these cells suggesting Fzd7 mediates non-canonical signalling of migrating colon cancer cells [Fzd7 also plays an important role during metastasis by regulating the critical stages in which epithelial cancer cells transition into mesenchymal cells when leaving the primary tumour (EMT), and then from mesenchymal to epithelial (MET) at the secondary site ,162,163.in vitro . MET in in vitro . On the er cells . Indeed,er cells .Furthermore, high-throughput screening for miRNAs implicated in human carcinomas has revealed significant downregulation of miR-23b in human colon cancers, which promotes metastasis, tumour growth, invasion and angiogenesis . SubsequBy far the most characterised cancer with relation to FZD7 is Hepatocellular carcinoma (HCC). Between 40%–70% of HCC display elevated nuclear β-catenin  suggestiFZD7 is frequently over expressed in HCC, especially in patients infected with HBV , and hasin vitro and in vivo, and was associated with inhibition of FZD7 [Post transcriptional control of FZD7 is also observed in HCC. The expression of miR199a is frequently downregulated in human liver tumours, and associated with poor clinical outcome. Experimental overexpression of miR199a in human HepG2 HCC cells was able to repress cancer growth  of FZD7 .FZD7 is overexpressed in the surrounding periportal and dysplastic liver suggesting it may be important during early stages of HCC . HoweverAPC is the 6th most frequently mutated gene in the non-hypermutated gastric tumours (over three times more frequent than the hypermutated subgroup which does not contain APC mutations). When combined with gain-of-function mutations in β-catenin, the Wnt pathway is found to be activated in over 10% of GCs [Nature Genetics in which the authors conclude that aberrant Wnt/β-catenin signalling plays an important role in gastric adenoma [in vivo [Wnt signalling is emerging as an important oncogenic pathway in gastric cancer (GC). The recent Cancer Genome Atlas paper, which analysed 295 primary gastric adenocarcinomas, identified that % of GCs . These o adenoma . Deregul[in vivo .in vitro tumourgenicity and tumour xenograft burden of Wnt pathway-activated gastric cancer models by means of attenuating Wnt/β-catenin signalling [Components that are known to be associated with signalling via FZD7 are strongly implicated in GC. For example, it has been shown extracellular secreted Wnt antagonists (sFRP and Dkk) are frequently epigenetically silenced through promoter hyper-methylation ,177. Crignalling ,177,178.There are several reports suggesting that the non-canonical Wnt pathway is active in GC. RHOA, a downstream component of non-canonical Wnt signalling and FZD7 , is upreFzd7 is able to inhibit the growth of gastric cancer cells and in mouse models of GC and colony forming assays with human GC cells [Several reports demonstrate that FZD7 expression is upregulated in GC, with high expression associated with poor clinical outcome ,185,186.GC cells .H. pylori-induced gastric tumourigenesis, implicates a role for Fzd receptors to promote Wnt signalling in gastric cancer. As such, recent research has shown FZD7 to be highly expressed following H. pylori infection and associated increases in cell proliferation [FZD7 to be negatively regulated by miRNA-27b, which when overexpressed is sufficient to inhibit the expression of FZD7 and reduce gastric cancer cell proliferation. Likewise, targeted knockdown (siRNA) of FZD7 in H. pylori-induced gastric cancer cells effectively phenocopies the effect of miRNA-27b overexpression [Finally, the strong association between aberrant Wnt signaling and feration . Subsequpression .Wnt signalling regulates the development and homeostasis of the breast  and is dA screen to analyse which pathways were active in triple negative (TN) breast cancer, identified that the Wnt pathway was an important driver in this aggressive type of breast cancer and that FZD7 was often overexpressed . Indeed,+-dependent deacetylase positively regulates FZD7 mRNA and protein levels, promoting increased cell migration and proliferation [/+ΔNp63 MaSCs reveals alterations exclusively to Wnt signalling, with substantial decreases in Fzd7 and Wnt5a expression, resulting in a significant reduction in MaSCs [FZD7 enhancer in p63 bound chromatin, which was further supported by the activation of a FZD7 enhancer-driven luciferase reporter in response to ΔNp63, thus demonstrating Fzd7 is a direct target of ΔNp63 [ΔNp63 or FZD7 in patient-derived xenografts (PDX) from TN breast tumours significantly attenuated tumourigenicity, which was associated with a reduction in the number of TICs [Furthermore, recent research has revealed a novel mechanism by which FZD7 expression is regulated in breast cancer cells. SIRT1, an NADferation . As suchferation . In addiferation . Gene-sein MaSCs . Chromatof ΔNp63 . Subsequ of TICs . These dAs with other cancers, a role for the Wnt signalling pathway at the level of the receptor/ligand has been suggested from studies on the SFRP genes. SFRP1 SFRP2 and SFRP5 are methylated in ovarian cancer , and expOvarian cancer is a heterogenous disease but can be broadly separated into five distinct subtypes based on molecular characteristics; epithelial-A (Epi-A), Epi-B, mesenchymal (Mes), Stem-A and Stem-B. FZD7 is upregulated in the Stem-A subgroup of ovarian cancer, which has a poor clinical outcome . KnockdoInterestingly, inhibition of FZD7 in ovarian cancer cells resulted in an increase in the level of canonical signalling, whilst the non-canonical signalling components including RhoA were slightly down regulated. These data suggest FZD7 maybe regulating ovarian cancer cell growth via the Wnt/PCP pathway rather than the canonical pathway, which is upregulated in Stem-A ovarian tumours .APC and β-catenin are rare in this cancer, suggesting that Wnt signalling could be activated at the level of the receptor in cervical cancer [Nuclear β-catenin is observed in ~70% of cervical tumours indicating deregulation of Wnt signalling is common in cervical cancer . Howeverl cancer . Indeed,l cancer , WIF-1 [l cancer , SFRP-1,l cancer  in cerviin vitro [FZD7 to be a direct target of miRNA-142-3p, which is normally expressed at low levels in cervical cancer cells. Importantly, restoration of miRNA-142-3p expression significantly reduced FZD7 expression, cell proliferation and invasion, thus identifying miRNA-142-3p and FZD7 as attractive therapeutic targets for cervical cancer therapy [Inhibition of FZD7 using shRNA was able substantially reduce invasion and EMT in HeLa and SiHa cervical cancer cell lines in vitro . These pin vitro . Moreove therapy .There is no data to demonstrate that FZD7 is upregulated in cervical cancer, however, as mentioned above, the role of FZD7 in cancer could be underestimated, as seems the case in cervical cancer, due to changes in the membrane turnover of FZD7, which will greatly affect the response of a cell to Wnt ligands.2+ pathway [There are conflicting reports on the role of canonical Wnt signalling in melanoma. Some reports have shown that canonical Wnt signalling is reduced as melanoma progresses, suggesting that activating canonical Wnt signalling could be a therapeutic strategy for treatment of melanoma. Indeed, overexpression of Wnt3a reduced proliferation of human or mouse melanoma cells , and pha pathway , and as  pathway ,209.FZD7 is upregulated in human, metastatic melanoma cells, and shRNA mediated knockdown of FZD7 in these cells was able to inhibit formation of xenograft tumours and also metastasis following intravenous injection . MelanomWilm’s tumour (WT) is a renal solid tumour, most commonly observed in children. Canonical Wnt signalling is frequently deregulated in Wilm’s tumour, associated with mutations to β-catenin , and FZDInhibition of FZD7 using a blocking antibody resulted in reduced clonality of cancer cells isolated from human WT samples, and apoptosis in those cells which expressed FZD7. Interestingly, WT cells become resistant to treatment with FZD7 antibody when SFRP1 or DKK1 are expressed, suggesting that the Wnt pathway is deregulated at the level of the Wnt receptor complex in WT .Apc in the kidneys of mice resulted in multiple renal tumours, indicating that deregulated canonical Wnt signalling is sufficient to induce tumourigenesis in the adult kidney [In renal cell carcinoma (RCC), Wnt signalling is also activated, with reports of loss of Apc  and mutat kidney .in vitro, whilst inhibition of FZD7 with shRNA is able to reduce proliferation in the human RCC cells [Deregulation of Wnt signalling at the level of the Wnt receptor is also implanted in RCC as SFRP1, SFRP2, SFRP4, SFRP5, DKK3, and WIF1 are hypermethylated in primary RCC samples . FZD7 haCC cells , suggestDeregulated Wnt signalling is observed in many cancers, and, as described above, the Wnt receptor complex is often involved in this aberrant Wnt activation. Although the role of FZD7 has not been described in all cancers with high Wnt activity it is tempting to speculate that FZD7 may be involved in these cancers also, particularly as it is a Wnt target gene ,88, and FZD7 is upregulated in esophageal cancer [FZD7 is upregulated in nasopharyngeal carcinoma [FZD7 is upregulated in human glioblastoma stem cells, and whilst no experiments have yet been performed to specifically address the role of FZD7 in these cells, overexpression of sFRP1 halts cell cycle and induces apoptosis, suggesting the Wnt receptor complex, and possibly FZD7, are attractive targets to treat glioblastoma [l cancer , but thearcinoma ,220, andarcinoma , but thearcinoma ,223 and arcinoma ,225, butarcinoma . FZD7 isblastoma ,228.Wnt signalling does not act alone in promoting cancer, and is able to co-operate with other pathways, oncogenes and tumour suppressors, including JAK/STAT , K-ras ,230, p53Fzd7 was deleted, but eventually recovered and was associated with an increase in the expression of Fzd2, suggesting Fzd2 may have been able to compensate for Fzd7 loss in this setting [The plethora of studies detailed above has established that signalling via Wnt/FZD is epigenetically regulated in many cancers, and therefore offers an attractive target for therapeutic intervention. As FZD receptors belong to a large family of proteins with high levels of promiscuity to several Wnt ligands it was initially thought that targeting a single FZD receptor would not be an effective strategy to treat cancer due to functional redundancy by another FZD receptor. However, this has not proven to be the case, as experiments inhibiting FZD7 alone have shown that it can have potent anti-tumour effects, as detailed above. Interestingly, DNA damage induced regeneration in the intestine was impaired when  setting . It willApc do not completely prevent its action to regulate β-catenin and thus upstream events at the level of the receptor can also be targeted in cancers with mutant Apc [Apc is mutated at several different places, with different levels of Wnt pathway activity associated with each mutation, most likely as a result of the mutations ability to prevent Apc regulating β-catenin phosphorylation. Thus, all Apc mutations do not result in the same impact on Wnt signalling, and it will be useful in future research to map which Apc mutations correlate to a cellular response to upstream regulators at the level of the ligand/receptor. This information will be important to help stratify which patients will respond to cancer treatments targeting FZD7 or any other regulators of the ligand/receptor complex, based on which mutations to Apc their individual cancers have.Deregulated Wnt signalling in cancer is often the result of mutations to cytoplasmic components of the Wnt pathway. Although the concept of targeting a receptor for a cancer driven by a mutation to a downstream, cytoplasm signal transducer (such as Apc), seems counter-intuitive, the Goldilocks model of Wnt signalling helps explain how this is possible. The data that has contributed to this model demonstrates that mutations to tant Apc . Apc is Fzd7 was deleterious to intestinal stem cells, the intestine has a remarkable capacity to repopulate the epithelium with non-recombined, Fzd7 proficient cells, and therefore inhibition of Fzd7 is well tolerated. Thus, future tumour prevention strategies could employ inhibition of FZD7 to prevent Wnt induced transformation of stem cells in FAP patients who are inherently predisposed to develop this type of pathology due to loss of heterozygosity of APC. Cancer stem cells are biologically distinct from the differentiated cancer cells that form the bulk of a tumour, and are often the cells responsible for the continued proliferation and resistance to therapies of the tumour [FZD7, but not FZD2 or FZD1, expression is dramatically up-regulated in CRC [Stem cells are required for the continuous tissue maintenance within diverse organs, and Wnt signalling has been identified as regulating stem cells in several organs including the gastrointestinal tract, breast, skin, kidney, and ovary . Stem cee tumour . Lgr5 ald in CRC , thus cod in CRC .Another potential strategy for the use of FZD7 inhibition could be to target disseminated metastatic cells that are responsible for the development of secondary tumours, often many years after the primary tumour has been successfully removed by surgery/chemotherapy. Dormant, disseminated tumour cells are the hindrance to curative treatment for colon cancer, the vast majority of deaths from colon cancer attributable to tumour recurrence. CRC can metastasise early, long before diagnosis, and the disseminated cells can sit dormant for many years, providing ample opportunity to target these cells and eliminate them . As detaFzd7 is able to significantly reduce the transcription of Wnt target genes suggesting that such an antibody would be effective in combating specific cancers [As FZD7 is located on the cell surface, this enables it to be targeted efficiently using antibodies. Indeed, monoclonal antibodies to target FZD7 developed by Oncomed Pharmaceuticals (OMP-18R5 or Vantictumab) are already in phase 1b clinical trials for solid tumours including colorectal, neuroendocrine, breast, sarcoma, pancreatic and NSCLC . As Wnt  cancers .APC can respond to FZD7 inhibition. The mechanism for this is not yet fully understood, but could be dependent on the type of mutation present in Apc, or the ability of FZD7 to transmit signalling via all the currently known Wnt pathways and consequently by-pass Apc. Thus, FZD7 acts as the lynchpin for multiple Wnt transduction pathways, depending on the availability of co-receptors, ligands and antagonists. This promiscuity of FZD7, may represent the Achilles’ heel of targeting the Wnt receptor complex for the treatment of cancer, as FZD7 is involved in multiple malignancies characterised by deregulated Wnt signalling.Over recent years it has become clear that activation of the Wnt pathway at the level of the receptor, via upregulation of Wnt ligands and downregulation of Wnt receptor antagonists, plays a major role in promoting cancer initiation, growth and metastasis. Even cancers which contain mutations to cytoplamsic transduction components such as


Six binding elements for Wnt/β-catenin signalling transcription factor binding elements (T-cell factor/ lymphoid enhancing factor) were identified in the human ALDH1B1 gene promoter (3 kb) but shown by dual luciferase reporter assay to not be necessary for ALDH1B1 mRNA expression in colon adenocarcinoma cell lines. We examined Wnt-reporter activity and protein/mRNA expression for Wnt, Notch and PI3K/Akt signaling pathways. Wnt/β-catenin, Notch and PI3K/Akt-signaling pathways were down-regulated in SW 480 cells in which ALDH1B1 expression had been suppressed. In summary, our data demonstrate that ALDH1B1 may promote colon cancer tumorigenesis by modulating the Wnt/β-catenin, Notch and PI3K/Akt signaling pathways. Selective targeting of ALDH1B1 may represent a novel means to prevent or treat colon cancer.In the normal human colon, aldehyde dehydrogenase 1B1 (ALDH1B1) is expressed only at the crypt base, along with stem cells. It is also highly expressed in the human colonic adenocarcinomas. This pattern of expression corresponds closely to that observed for Wnt/β-catenin signaling activity. The present study examines the role of ALDH1B1 in colon tumorigenesis and signalling pathways mediating its effects. In a 3-dimensional spheroid growth model and a nude mouse xenograft tumor model, shRNA-induced suppression of ALDH1B1 expression decreased the number and size of spheroids formed  All samples were incubated at 37°C for 45 min and then centrifuged and washed with 500 μl Aldefluor buffer. ALDH+ cells were evaluated by the University of Colorado Cancer Center flow cytometry core facility.Cell count and viability of scramble and ALDH1B1 shRNA-transfected SW480 cells were determined on a Countess Automated Cell Counter (Life Technologies). Cells in both groups exhibited more than 80% viability. The Aldefluor reagent system  was used to detect intracellular catalytic activity of ALDH. In brief, cells were centrifuged (250 G for 5 min) and pellets were resuspended in Aldefluor assay buffer containing 15 μM BAAA at a concentration of 10All quantitative experiments were performed at least in triplicate, and the data are presented as the means ± S.E. Statistical significance was determined by Student’s unpaired t-test or one-way ANOVA using SigmaStat Statistical Analysis software . P < 0.05 was considered to be significant.Apc(+/+)) mice expressed immunopositivity for ALDH1B1 in a small subset of the columnar cells at the bottom of crypt  and in some cells along the side of the lower middle part of the crypt . Six binding sites for TCF/LEF transcription factors were identified in the 3000 bp upstream sequence from transcription start site of the ALDH1B1 gene, which comprise a minimum element of 5′-(A/T)(A/T)CAA(A/T)G-3′  program (A/T)G-3′  31]. TB. TBwww.cA/T)G-3′ .ALDH1B1 and c-Myc in these colon cancer cells. ALDH1B1 promoter activity was 2-, 17- and 233-fold that of the control plasmid in the HT29, SW480 and COLO320 cells, respectively. A similar cell line order of activity was observed for the c-Myc promoter activity . Since these cells exhibited high levels of ALDH1B1, we transfected the SW480, COLO320 and HT29 cell lines with ALDH1B1-pGL3 or cMyc-pBV luciferase vector and used corresponding empty plasmids as a control. Luciferase activity was measured to determine the basal level of activation of the promoters for activity . These ractivity . We alsoWe used the SW480 cell line to examine the effect of suppressing ALDH1B1 expression on tumor growth. This was accomplished by shRNA-mediated knockdown. The effectiveness of shRNA knockdown was evaluated by Western blotting and ALDH1B1 expression was decreased in the ALDH1B1 shRNA cells by 80% .in vitro three-dimensional culture system and an in vivo xenograft model. Cells from the scramble (control) and ALDH1B1 knockdown groups were grown in three-dimensional culture to examine spheroid formation. After 10 d of incubation, the volume and number of spheroids were lower in the ALDH1B1 knockdown cell cultures than in control cell cultures  SW480 cells contain a small population of cells (~ 3%) with very high ALDH catalytic activity, so-called ALDHht cells . The capht cells  is consibout 90% . Our finApcMin mice, we examined the effect of ALDH1B1 knockdown on Wnt/β-catenin signaling. We employed the TOPFlash/FOPFlash Wnt activity reporter assay using scramble (control) shRNA and ALDH1B1 knockdown SW480 cells. Decreased β-catenin-mediated transcriptional activity occurred in SW480 ALDH1B1 knockdown cells 24 h after shRNA transfection , c-Myc and LGR5. RT-PCR analysis showed a strong induction of Axin2 and suppression of CTNNB1, c-Myc and LGR5 expression in the ALDH1B1 knockdown SW480 cells , including high tumorigenicity, self-renewal and chemoresistance [bright cells, showing that ALDH1B1 was responsible for the high ALDH activity of ALDHbright SW480 colon cancer cells. Given that ALDHbright cells are considered to possess high tumorigenic potential [High levels of ALDH1B1 in human colon cancer cells are suggestive of a role for ALDH1B1 in colon tumorigenesis. To examine this possibility, we used an shRNA approach to suppress or knock down expression of ALDH1B1 in SW480 cells, a human colon cancer cell line. The shRNA transfection caused an 80% reduction in ALDH1B1 levels. We selected SW480 cells for tumor formation assays because they exhibit high constitutive expression of ALDH1B1 and very low constitutive ALDH1A1 expression . Hence, any possible obfuscating effects of ALDH1A1 on tumorigenicity would be minimized. Suppression of ALDH1B1 expression by shRNA reduced spheroid formation by SW480 cells in a three-dimensional cancer cell culture and reduced tumor growth by SW480 cells in an er cells –38. Howeer cells ,39. Cellsistance ,40. Knocotential , the resbright SW480 colon cancer cells, we examined the relationship between ALDH1B1 and the Wnt/β-catenin signaling pathway. First, we hypothesized that ALDH1B1 may be regulated by Wnt/β-catenin signaling. Since activation of Wnt-signaling is mediated through binding of β-catenin to TCF/LEF transcription factors, the presence of TBE is required in the promoter regions of all the genes under direct control of the Wnt/β-catenin signaling pathway [ALDH1B1 3kb promoter region, making it a potential target for control by β-catenin and, by extension, the Wnt/β-catenin signaling pathway [ALDH1B1 promoter was transcriptionally active in colon cancer cell lines. However, even after sequential deletion of all six TBE sites from the ALDH1B1-pGL3 promoter construct, promoter activity remained the same. This indicates that none of the potential TBE sites found within the 3 kb promoter region of ALDH1B1 are contributing to the transcriptional activation of the ALDH1B1 promoter. In SW480 cells, deletion of the promoter region containing TBE2 to TBE6 resulted in increased ALDH1B1 promoter activity, suggesting that transcription repression factors are present in this region of the promoter and are functional in these cells. Given that all of the examined cell lines showed ALDH1B1 promoter activity even after deletion of all the six putative TBEs, it may be speculated that the remaining promoter region (-492/+115 bp) has some binding elements for transcriptional activators. We explored this possibility and found binding sites for various transcription factors known to be important in colon cancer [Axin2 mRNA and suppression of CTNNB1, c-Myc and LGR5 mRNAs. Axin2 negatively regulates Wnt/β-catenin signaling and mutations in Axin2 have been described to be oncogenic in colorectal cancer [C-Myc is an oncogene and downstream target of Wnt/β-catenin signaling [Several inherited and sporadic colorectal cancer studies have established that Wnt/β-catenin signaling plays a critical role in colon tumorigenesis . With ou pathway –44. In t pathway . In addin cancer –50. Furtl cancer ,52. C-Myignaling  and overignaling . Downregignaling . LGR5 isignaling ,56. CollApcMin mice, which have mutated Apc gene and resultant hyperactive Wnt signaling, show enhanced Notch signaling activity [Notch/ApcMin double mutant mice. These mice exhibited an increase in the number of intestinal tumors and dysplastic lesions in the colon relative to the ApcMin mice [Overexpression of Notch-related proteins in cells with active Wnt signaling at the bottom of intestinal crypt is suggestive of cross-talk between Wnt and Notch signaling ,26. In aactivity . Overexpactivity . This inactivity . The synMin mice . In addiMin mice . Our resMin mice , suggestin vitro and in vivo tumor formation assays. Our results show that PI3K/Akt pathway is down-regulated upon ALDH1B1 suppression, possibly by down-regulating PPARβ/δ which corroborates earlier findings and suggests an association between this pathway and colorectal cancers [The mechanism by which ALDH activity promotes tumor growth is the subject of intensive research. Retinoic acid (RA) has been reported to increase expression of pro-survival genes and tumor growth by activating PPARβ/δ in colon cancer in cells with high FABP5 relative to CARBPII, which in turn can modulate PI3K/Akt pathway ,59. This cancers . A summaOur findings demonstrate that ALDH1B1 may not only be a potential biomarker for colon cancer but also play a functional role in the formation of tumors in colorectal cancer. In addition, the present study provides mechanistic insights into the means by which ALDH1B1 may promote tumor formation, i.e., by modulating the Wnt/β-catenin, Notch, and PI3K/Akt signaling pathways. Finally, it is apparent that ALDH1B1 can be a significant contributor to ALDH activity measured by the Aldefluor assay. These results substantiate the accumulating evidence that ALDH isozymes other than ALDH1A1 may contribute to the cancer stem cell phenotype identified by the Aldefluor assay . As such


Rspo1/2/3/4) in the hair follicle dermal papilla is revealed. Expression of Rspo1 in the dermal papilla is specifically and prominently upregulated before anagen entry, and exogenous recombinant R-spondin1 protein injection in mid-telogen leads to precocious anagen entry. Moreover, R-spondin1 activates Wnt/β-catenin signaling in cultured bulge stem cells in vitro, changing their fate determination without altering the cell proliferation. Our pioneering study uncovers a role of R-spondin1 in the activation of cultured hair follicle stem cells and the regulation of hair cycle progression, shedding new light on the governance of Wnt/β-catenin signaling in skin biology and providing helpful clues for future treatment of hair follicle disorders.R-spondin proteins are novel Wnt/β-catenin agonists, which signal through their receptors leucine-rich repeat-containing G-protein coupled receptor (LGR) 4/5/6 and substantially enhance Wnt/β-catenin activity. R-spondins are reported to function in embryonic development. They also play important roles in stem cell functions in adult tissues, such as the intestine and mammary glands, which largely rely on Wnt/β-catenin signaling. However, in the skin epithelium and hair follicles, the information about R-spondins is deficient, although the expressions and functions of their receptors, LGR4/5/6, have already been studied in detail. In the present study, highly-enriched expression of the R-spondin family genes ( The Wnt/β-catenin signaling is accepted as the dominant player regulating multiple aspects of skin biology, such as the morphogenesis and patterning of skin epithelium and its appendages, the self-renewal and differentiation of resident stem cells in skin, as well as the functions of numerous differentiated skin cells . Hair foThe newly-discovered R-spondin1-4 is a small family of vertebrate-specific secreted growth factors that strongly enhance Wnt/β-catenin activity by collaborating with their receptor LGR4/5/6 ,8 and thSeveral studies have revealed the implication of R-spondins in the skin. In particular, R-spondin2 is reported to be associated with hair coat characteristics in dogs . InteresRspo1/2/3/4) were highly expressed in the hair follicle dermal papilla, and the expression of Rspo1 prominently was upregulated before anagen entry. In vivo, the precocious anagen entry happened because of the exogenous recombinant R-spondin1 protein injection. In vitro, R-spondin1 activated Wnt/β-catenin signaling in cultured bulge stem cells, changing their fate determination without altering the cell proliferation. In conclusion, our study demonstrated an unrecognized role of R-spondin1 mediating between the DP and the hair follicle to initiate regenerative hair cycling.Herein, we examined the spatiotemporal expressions of R-spondins in the telogen hair follicles and revealed especially the role of R-spondin1 in the induction of anagen and the regulation of HFSC activation. R-spondin family genes  fluorescence and the Lef1+ DP was specifically labeled with red fluorescent protein (RFP) fluorescence  activity  and immune cell marker Ptprc (CD45 gene). Therefore, the vast majority of K14−/Lef1+ cells we sorted were DP cells.To determine whether R-spondins function in anagen initiation, we first located the expression of R-spondin genes in different cell populations in the telogen mouse skin. K14-H2B-GFP/Lef1-RFP double transgenic mice were used to isolate epidermal cells, dermal fibroblasts and DP cells . In telorescence a. By flurescence b: the K1−/Lef1+ cells, ΔCRspo1t (CRspo1t − CGapdht) was 3.33 ± 0.56; ΔCRspo2t was 6.81 ± 1.47; ΔCRspo3t was 8.82 ± 0.80; ΔCRspo4t was 4.63 ± 0.85. It is well-known that DP signals are important in regulating HFSC activation to initiate anagen. To determinate the correlation of R-spondin genes in DP cells and the hair cycle, we next investigated the dynamic expression changes of these genes during telogen. After cell populations sorted as indicated in Rspo2 and Rspo3 mRNA levels were relatively stable, Rspo1 and Rspo4 mRNA levels showed significant upregulation in late telogen  staining and anti-R-spondin1 antibody immunoblotting. The bands for R-spondin1-Fc protein were detected around 60 KDa  mice for one week according to a schedule shown in The advanced appearance of new hairs at the injection site indicated a precocious anagen entry, suggesting that R-spondin1 promoted the induction of anagen in hair follicles. To study the precocious anagen entry in detail, we also performed the injection experiments on TOP-Gal mice, in which the activation of Wnt/β-catenin could be monitored through the specific X-gal staining. There were all-telogen hair follicles and no X-gal staining in TOP-Gal mice on PD55 (the time before injection) f. One wein vitro. The hair follicle bulge stem cells we used were originally derived from the K14-H2B-GFP mice based on GFP and CD34 expression as previously described [Lgr4 was detected in the cultured bulge stem cells by RT-PCR, whereas Lgr5 and Lgr6 were absent.One inevitable event of the anagen entry is the HFSC activation. Since R-spondin1 injection can trigger the earlier anagen entry in the hair cycle, the effect of R-spondin1 on HFSCs was further investigated escribed . The celAxin2 expression was elevated by the treatments of R-spondin1 or Wnt3a + R-spondin1 whereas Myc expression was not altered. Similarly, Lef1 and Tcf7 expression was also upregulated. Interestingly, Lgr5 upregulation was prominent by R-spondin1 supplementation whereas Wnt3a alone showed no effect at all. We also observed a very subtle augment in Cyclin D1 (Ccnd1) mRNA level in R-spondin1 and Wnt3a + R-spondin1 groups, suggesting possible regulation of HFSC proliferation by R-spondin1.Given the expression of R-spondin receptor LGR4 in the bulge stem cells, these cells were starved and treated with Wnt3a and/or R-spondin1, and then the activation of Wnt/β-catenin signaling was studied. Western blotting results showed an obvious activation of β-catenin after the addition of either Wnt3a+R-spondin1 or R-spondin1 alone d, whereain vitro, we assessed the expression of genes that are associated with cell proliferation, differentiation, and hair follicle fate determination. Many of these genes are also reported to be Wnt/β-catenin targets. We first examined genes involved in cell cycle regulation, quiescence maintenance, and cell proliferation, including cyclins (Ccnb1/2/3), cyclin-dependent kinase inhibitors (Cdkn1a/1b/1c) and Mki67  [Rspo2) [Rspo2) [Rspo2) [etc., and proved to regulate the morphogenesis of these tissues [Rspo1 and Rspo4 expression exhibited significant upregulation right before anagen entry when compared with early-mid telogen. The increase in Rspo1 expression is consistent with the previous microarray data in which DP gene expression on late telogen (PD69) and early telogen (PD43) were compared [Rspo1/4 expression in DP. Furthermore, the role of R-spondin4 in regulation of the hair cycle need to be further studied.The expression of R-spondins in the skin was occasionally reported. In an earlier study, it was mentioned that expression of R-spondin1 was seen in patches of the developing mouse dermis at E12.5 and then restricted in the DP since E18.5 . Rspo3 aat E14.5 . Studiesinocytes ,26. The  [Rspo1) , the bra [Rspo2) , the tee [Rspo2) , the lun [Rspo2) , etc., a tissues ,30. In tcompared . Intereset al. [Rspo1 knockout would help address this question.During telogen, the hair follicle is kept in a resting status mainly by bone morphogenetic protein (BMP) signaling, and the antagonistic interplay between BMP, Wnt/β-catenin, and some other signaling, such as TGF-β, regulates the choice for HFSC activation and anagen entry . Althouget al. . Neverthin vitro to help find possible mechanism(s) through which exogenous R-spondin1 injection promote anagen entry. It is reported that LGR5 is strongly expressed in the lower portion of the CD34+ bulge stem cells in telogen [in vitro assays which was originally derived from telogen mouse back skin based on CD34 expression [in vitro. Nevertheless, these bulge stem cells express another R-spondin1 receptor, LGR4, which, we believe, makes them responsive to R-spondin1. In our in vitro study, R-spondin1 supplementation alone exhibits a considerable effect on the enhancement of Wnt/β-catenin activity, indicating the existence of endogenous Wnt ligands. Indeed, we detected there were the expression of several Wnt ligands, either in bulge stem cells or in the feeder cells, such as Wnt3a, Wnt4, Wnt5, and Wnt10a. Therefore, we pondered R-spondin1 might exaggerate the Wnt/β-catenin activity in the presence of some of these Wnt ligands.We further studied the effect of R-spondin1 on bulge stem cells  telogen , but thepression  did not in vitro, such as Axin2 and Lef1, are reported to be associated with HFSC activation in vivo upon telogen-to-anagen transition [per se. Intriguingly, as mentioned above, LGR5 was initially absent in the bulge stem cells in vitro, but it showed a drastic upregulation with R-spondin1 treatment , implying the potential importance of R-spondin1 in HFSC activation. We did not detect evident changes in the expressions of genes associated with cell proliferation , but substantial change in HFSC fate related genes which are also Wnt/β-catenin targets  occurred. Based on these data, it is speculated that R-spondin1-mediated Wnt/β-catenin activation does not directly regulate HFSC proliferation in vitro, but possibly control HFSC fate determination and in turn initiates HFSC activation.Several of the Wnt target genes that were upregulated by R-spondin1 (or together with Wnt3a) in the bulge stem cells ansition . LGR5, wRspo1 expression level upon telogen-to-anagen transition. Exogenous R-spondin1 protein injection in the mid-telogen induces precocious anagen entry in the hair follicle. Moreover, R-spondin1 protein activates Wnt/β-catenin in HFSCs in vitro, possibly affecting HFSC fate determination, but does not alter HFSC proliferation. Given the central role of Wnt/β-catenin in the homeostasis and regeneration of the skin and the hair follicles, our study provides helpful clues for the future treatment of disorders such as alopecia.In conclusion, we herein reveal highly enriched expression of R-spondin genes in the hair follicle DP in the mouse back skin and a specific increase in LacZ transgene, and primers “CTAGGCCACAGAATTGAAAGATCT” and “GTAGGTGGAAATTCTAGCATCATCC” were used to amplify the internal control. Wild-type CD1 mice were purchased from the Vital River Company .The K14-H2B-GFP/Lef1-RFP double transgenic mice were a kind gift from Elaine Fuchs’ lab and were originally derived as previously described . The TOPMice were maintained under specific-pathogen-free (SPF) conditions with a constant photoperiod (12L:12D) and free access to food and water. All mice were bred and used according to the “Guidelines for the Care and Use of Animals in Research” of the Institute of Zoology, Chinese Academy of Sciences, and all experiments which involved the use of the mice were approved by the ethics committee of the Institute of Zoology, Chinese Academy of Sciences .−/RFP+, GFP−/RFP−, and GFP+/RFP−) were sorted from each individual mouse dorsal skin sample in a BD Aria Flow cytometer . Sorted cells were then either cytospinned for immunostaining or centrifuged for RNA extraction.The K14-H2B-GFP/Lef1-RFP mice were euthanatized at indicated postnatal days. Individual mouse dorsal skin was shaved and cut off, after the subcutaneous fat gently removed, the skin was cut into very small pieces and subsequently incubated with 2.5 mg/mL Type IV collagenase  in a shaker for 4 h at 37 °C until there was no other solid matter remaining but hair shafts. Single cell suspension was obtained by pipetting, straining, repeated washing, and centrifugation. Three different cell populations   for 10 min at room temperature. After washed with PBS for three times, samples were incubated in 5% BSA and 0.2% triton-X 100 (Sigma) in PBS for one hour at room temperature, and then incubated with primary antibodies at 4 °C overnight. For detection, FITC- or TRITC-conjugated secondary antibodies  were added for one hour at room temperature. After samples were washed with PBS, propidium iodide (PI) or Hoechst 33342 (both form Sigma) was used for nuclear staining. All images were acquired with Nikon Eclipse 80i microscope . The following primary antibodies were used: rat anti-CD45  and rabbit anti-CD117 .Cells on coverslip were fixed with 4% PFA, then incubated with NBT-BCIP solution (Sigma) for 30 min at room temperature. After washed with PBS, the cells were counterstained with nuclear fast red  and dehydrated with alcohol  gradient followed by xylene , then coverslipped with a resinous mounting medium .® F Affinity Chromatography Sorbent , and concentrated in saline using the 10 K Amicon-Ultra Centrifugal Filter . The yield of the purified protein was determined with a BCA Protein Quantification Assay Kit . The level of R-spondin1-Fc protein was verified by CBB staining and immunoblotting with the R-spondin1 antibody .Stably-transfected 293T cells producing mouse R-spondin1-Fc fusion protein was a kind gift from Calvin Kuo at Stanford University. This fusion protein contains a C-terminal mouse IgG2-Fc fragment and was purified from the conditioned medium of the R-spondin1-293T cells via protein A affinity chromatography . BrieflyThe TCF-luciferase assay was performed as previously described . Brieflyn = 5 for the control group; n = 6 for the R-spondin1-Fc group; n = 4 for the Noggin group).Telogen CD1 or TOP-Gal mice were injected with R-spondin1-Fc fusion protein according to a schedule shown in 2 and then incubated overnight in staining buffer 3, 5 mM K4Fe(CN)6, 2 mM MgCl2, and 0.1 M phosphate buffer, all from Sigma, at 37 °C in the dark. 70% alcohol was used to stop the reaction and store the stained tissue samples. For histological observation, samples were dehydrated through a gradient of ethanol, embedded in paraffin , and then cut into 10–20 μm-thick sections using the CM1950 platform (Leica Instruments). The tissue sections were deparaffinized, rehydrated, and then counterstained with eosin .β-galactosidase (LacZ) staining were performed as previously described . BrieflyMouse H2B-GFP bulge cells were a kind gift from Ting Chen’s lab at the National Institute of Biological Sciences, Beijing. Cells were cultured and expanded on NIH-3T3 mouse fibroblast feeder cells treated with mitomycin C (Sigma) as described previously . For sti® AQueous One Solution Cell Proliferation Assay (MTS) Kit (Promega) following the manufacturer’s instructions. Absorbance at 490 nm was examined in a BioTek Microplate Reader (BioTek Instruments Inc.). For statistical analysis, every group had a triplicate. Data were normalized to the control group. Data are presented as means ± SEM. and differences were considered significant with a p value less than 0.05.Bulge cells were cultured and treated as described above. The cell proliferation assay was performed using CellTiter 96® Reverse Transcription System (Promega). Semi-quantitative PCR (qPCR) was performed using GoTaq® qPCR Master Mix (SYBR Green) (Promega). The qPCR procedure was 94 °C for 2 min and 45 cycles of 95 °C for 15 s, 55 °C for 15 s, and 68 °C for 25 s. PCR was performed using Promega PCR Master Mix (Promega) and the procedure was 94 °C for 5 min; 24 (only for Gapdh) or 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; 72 °C for 5 min. The primers used in PCR are listed in Gapdh expression was calculated using the ΔΔCt method with efficiency correlation [t-test. Data are presented as means ± SEM. and differences were considered significant with a p value less than 0.05.Different cell populations sorted by FACS were centrifuged and the total RNA was extracted using a ReliaPrep™ RNA Cell Miniprep System (Promega). cDNA was generated with a GoScriptrelation . No lesst-test. Differences were considered significant with a p value less than 0.05.The Western blotting protocol was described in detail previously . BrieflyThe following primary antibodies were used: mouse anti-active β-catenin , mouse anti-β-catenin , rabbit anti-Axin2 , mouse anti-MYC , rabbit anti-phospho-GSK3β (Ser9) , rabbit anti-GSK3β , and rabbit anti-GAPDH .


It is well established that plant phenolics elicit various biological activities, with positive effects on health. Palm oil production results in large volumes of aqueous by-products containing phenolics. In the present study, we describe the effects of oil palm phenolics (OPP) on several degenerative conditions using various animal models. OPP reduced blood pressure in a NO-deficient rat model, protected against ischaemia-induced cardiac arrhythmia in rats and reduced plaque formation in rabbits fed an atherogenic diet. In Nile rats, a spontaneous model of the metabolic syndrome and type 2 diabetes, OPP protected against multiple aspects of the syndrome and diabetes progression. In tumour-inoculated mice, OPP protected against cancer progression. Microarray studies on the tumours showed differential transcriptome profiles that suggest anti-tumour molecular mechanisms involved in OPP action. Thus, initial studies suggest that OPP may have potential against several chronic disease outcomes in mammals. Phenolic phytochemicals are secondary metabolites of plant origin. These metabolites protect the plants against biological and environmental stresses. Because of their important protective biological functions, they are ubiquitous in plants and therefore are found in almost all food groups. Despite their wide distribution, the health effects of dietary phenolics have only recently attracted the interest of nutritionists, more attention having been paid before to antioxidant vitamins such as ascorbic acid, tocopherols and carotenoids. The diversity and structural complexity of phenolics is one of the main factors that have delayed their research.. These beneficial effects are attributed to the antioxidant activity of the constituent phenolic metabolites. The phenolic ring and hydroxyl substituents of these compounds can function as effective antioxidants due to their ability to capture free radicals by donating hydrogen atoms or electrons. Their bioactivity may also be related to the ability to chelate metals and inhibit lipo-oxygenases. However, the biological effects of phenolics may extend well beyond the modulation of oxidative stress. For example, soya isoflavones modulate endocrine function by interacting with oestrogen receptors. NO bioavailability influences insulin-stimulated glucose uptake and vascular tone. There is scientific evidence that phenolics exert significant vascular protection because of their antioxidant properties and increased NO bioavailability. A detailed understanding of the molecular events underlying these various biological effects is essential for evaluation of the overall impact on disease risk and progression.Emerging epidemiological evidence is increasingly attesting to the positive effects of fruits and vegetables in managing chronic and infectious diseases,2. T. In the previous study, we showed that the vegetation liquor from the palm oil milling process is a rich source of phenolics and presented evidence based on in vitro and ex vivo studies that oil palm phenolics (OPP) have free radical-scavenging activity and protective bioactivities against CVD. In the present study, we describe in vivo whole animal studies designed to identify specific bioactivities in relation to chronic diseases. The studies provide an indication that OPP may provide protection against major degenerative disorders including CVD, diabetes and cancer.The importance and demand for natural antioxidants have grown in recent years. Fruit processing residues are attractive and inexpensive sources of phenolic antioxidants. For example, apple pomace has been reported to be a source of polyphenolic compounds,7. Iet al.. OPP contains numerous phenolic acids. There are three isomers of caffeoylshikimic acid that are found to be major components of the extract. Other phenolic acids include caffeic acid, protocatechuic acid and p-hydroxybenzoic acid. The detailed composition of OPP is as described earlier.For all the animal studies carried out in the present study, OPP was prepared according to the methods described by Sambanthamurthi The use of animals in the cardiovascular studies  and cardiac arrhythmia experiments) was approved by the Commonwealth Scientific and Industrial Research Organisation-Health Sciences and Nutrition Animal Experimentation Ethics Committee. All experimental procedures including the care, handling and maintenance of the experimental animals were performed according to the National Health and Medical Research Council guidelines for the use and care of animals for experimental purposes.NG-nitro-l-arginine methyl ester (l-NAME) that leads to an elevation in BP. In this experiment, 12-week-old male Sprague–Dawley rats  were fed a standard laboratory rat diet . OPP was provided as a drink (30 ml/rat per d) from the start of the experiment at two different concentrations: 1500 mg/l gallic acid equivalent (GAE) and 3000 mg/l GAE. After 4 weeks on OPP, rats were challenged with l-NAME , and the treatments were continued for a further 8 weeks. The four experimental groups included (1) control, (2) l-NAME, (3) l-NAME+OPP (1500 mg/l GAE) and (4) l-NAME+OPP (3000 mg/l GAE).An NO-deficient rat model of hypertension was used. This animal model involves the inhibition of endogenous NO production with . Briefly, pre-trained rats were placed in restraining tubes (IITC models 805 and 815), and the tails were occluded with an appropriate-sized cuff coupled to a pneumatic pulse transducer and electrophysiograph (IITC model 65-12). The pulse was detected as the cuff pressure was reduced, and the pressure at which the first pulse was detected was taken as the systolic BP and computed automatically (IITC software package). The ambient temperature was carefully controlled (30°C), and the average of three to four readings was taken as the final reading.BP was monitored fortnightly by a standard photoelectric tail-cuff procedure  as described previously,13. n 25), sourced from the Animal Resources Centre, were fed a pro-arrhythmic diet (consisted of a standard laboratory diet further supplemented with 5 % (w/w) lard; Glen Forrest Stock Feeders) for 4 months in the presence or absence of OPP. OPP was provided as the drinking fluid (35 ml/rat per d) at a concentration of 1500 mg/l GAE. The control group received water. At the end of the feeding period, animals were subjected to acute myocardial ischaemia for 30 min by a temporary occlusion of the left anterior descending coronary artery, and the vulnerability to ventricular tachycardia, ventricular fibrillation (VF) and sudden cardiac death was quantified.In this experiment, 4-week-old male Wistar Kyoto rats . The fat comprised 67·1 % SFA, primarily as lauric acid (12 : 0) and myristic acid (14 : 0). The control group was fed the atherogenic diet and provided distilled water as the sole drinking-water. OPP were tested at two concentrations in two groups of nine animals each, and these animals were fed the atherogenic diet and supplemented with OPP at a concentration of either 500 or 1000 mg/l GAE in the drinking fluid. At the end of the animal feeding, the rabbits were killed by exsanguination following anaesthetisation with a mixture of ketamine and zoletil (0·1 ml/kg body weight), and entire aortas were traced, dissected and fixed in 10 % (v/v) formalin. The aortas were then stained with Oil Red-O, which resulted in lipid deposits in the aortas being stained red. Delineation of atheromatous deposits in the intima and lesions were quantified using a digital image analysis system, as described by Paigen Arvicanthis niloticus), also known as African grass rats, from the Brandeis University breeding colony, were studied as the model of choice. In captivity, Nile rats fed a standard rat chow develop type 2 diabetes and exhibit all aspects of the metabolic syndrome examined to date. All animal experiments were conducted in accordance with protocols approved by the Animal Care Committee of Brandeis University.To examine OPP for possible protective effects against diabetes, Nile rats  were divided into two equal groups and studied for a 17-week period. The treatment group drank OPP at 1800 mg/l GAE as their water supply, whereas the untreated group received distilled water. A shorter pilot study indicated that treatment effects could be expected between 900 and 1800 mg/l GAE. The animals were weighed initially and before killing by exsanguination under anaesthesia at the end of the 17-week period.2+O2, 1:1 mixture) at study origin after the rats were deprived of food for 15 h overnight. Glucose was measured with an Elite XL glucometer  standardised against a conventional spectrophotometric assay.Fasting blood glucose was measured in tail blood following light anaesthesia . Insulin was determined using an ELISA kit for rat insulin .For terminal measurement of plasma TAG, total cholesterol (TC) and insulin, fasting blood samples were collected by cardiac puncture under O2 and redissolved in 1 ml of chloroform. An aliquot (10–20 μl) of each sample was dried under N2 and dissolved in 50 μl of Triton X-100 and chloroform (1:1 by volume). The solution was dried extensively to remove chloroform. TAG and TC were then determined using the appropriate Infinity™ kits.Liver lipids (TAG and TC) were extracted from 0·1 g of tissue ground with 4 g of sodium sulphate using a 2:1 chloroform–methanol solution. Total extract was combined and dried under Nn 15) and the other was given OPP (1500 mg/l GAE) (n 15). The mice were killed by exsanguination under anaesthesia after 4 weeks, when the presence of tumours was observed through palpation at the neck. Tumours were excised, weighed, their dimensions  measured, snap-frozen in liquid N2 and stored at − 80°C. Tumour volume was calculated with the formula π/6 × length × width × height, which has the highest correlation with tumour mass as this is the formula of an ellipsoid, which was the shape of the subcutaneous J558 tumours in the present study.In this experiment, 6-week-old male inbred BALB/c mice were purchased from the Institute of Medical Research, after ethical clearance on all experimental procedures involving animals was obtained from the Animal Care and Use Committee of the University of Malaya, Kuala Lumpur, Malaysia. The mice were injected subcutaneously at the dorsum of the neck with IgA-secreting J558 mouse myeloma cells (ATCC TIB-6) after acclimatisation for 1 week. The mice were then divided into two groups, one group was given distilled water  and QIAshredder homogeniser . The total RNA samples obtained were subjected to a NanoDrop 1000A Spectrophotometer for yield and purity assessment. Integrity of the total RNA samples was then assessed using the Agilent 2100 Bioanalyser  and Agilent RNA 6000 Nano Chip Assay Kit (Agilent Technologies). A total of four total RNA samples with the highest RNA integrity numbers and 28S/18S rRNA ratios within each condition were then selected for microarray studies.Amplification of total RNA samples, which were of high yield, purity and integrity, was carried out using the Illumina TotalPrep RNA Amplification Kit . The biotinylated-complementary RNA produced was then hybridised to the Illumina MouseRef-8 Expression BeadChip version 1  using the Direct Hybridisation Kit . Illumina MouseRef-8 Expression BeadChips contained 50-mer gene-specific probes for over 24 000 genes, which were designed based on the Mouse Exonic-Evidence-Based Oligonucleotide set, the RIKEN FANTOM 2 database and the National Center for Biotechnology Information RefSeq (Release 5) transcript database. Microarray hybridisation, washing and scanning were carried out according to the manufacturer's instructions. (accession no. GSE17614).In brief, complementary RNA was added to a hybridisation buffer, and the hybridisation mixture was then briefly heated and hybridised to an Illumina BeadChip. The hybridised microarray then underwent a series of washes using the wash buffers provided and 100 % (v/v) ethanol . Non-specific hybridisation was blocked before incubating the microarray with the Amersham Fluorolink Streptavidin Cy-3 dye  for detection, followed by a final wash with the wash buffer. The microarray was then dried and scanned with the Illumina BeadArray Reader confocal scanner and Illumina BeadScan software , available at the Malaysia Genome Institute, National University of Malaysia. The raw gene expression data obtained are available at Gene Expression Omnibus21  was then considered for further analysis. Gene expression values were normalised using the rank invariant method, and genes that had a detection level of more than 0·99 in either the control or treatment samples were considered significantly detected. To filter the data for genes that changed significantly in terms of statistics, the Illumina custom error model was used and genes were considered significantly changed at a ‘Differential Score’ of more than 13, which was equivalent to a P value of < 0·05.Quality control of the hybridisation, microarray data extraction and initial analysis were carried out using the Illumina BeadStudio software . Outlier samples were removed via hierarchical clustering analysis provided by the Illumina BeadStudio software and also using the TIGR MeV software 22, vi to ensure that the replicates of each condition were clustered to each other. The Euclidean distance metric and average linkage method were used to carry out the hierarchical clustering analysis.The genes and their corresponding data were then exported into the Microsoft Excel software  for further analysis. To calculate fold changes, an arbitrary value of 10 was given to expression values, which were less than 10. Fold changes were then calculated by dividing means of Signal Y (treatment) with means of Signal X (control) if the genes were up-regulated and vice versa if the genes were down-regulated. A two-way (gene and sample) hierarchical clustering of the significant genes was then performed using the TIGR MeV software22 to  and MAPPFinder software . The MAPPFinder software ranks GenMAPP (pathways) and gene ontologies based on the hypergeometric distribution. Readers are referred to Doniger et al. for further explanations of the terms used in the MAPPFinder software. GenMAPP and gene ontologies that had permuted P values of less than 0·05, number of genes changed of more than or equal to 2 and Z scores of more than 2 were considered significant. Boxes coloured yellow indicate genes that were up-regulated, while those coloured blue indicate genes that were down-regulated. Individual boxes that have different shadings within them indicate the presence of multiple probes (splice transcripts) within a single gene.Changes in biological pathways and gene ontologies were assessed via functional analysis, using GenMAPP23 andChanges in regulatory networks were also analysed through the use of the Ingenuity Pathways Analysis software . For each organ, a dataset containing differentially expressed genes and their corresponding fold changes was uploaded into the application. Analysis of up-regulated and down-regulated genes was carried out separately. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. These genes were then overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. Networks of these focus genes were then algorithmically generated based on their connectivity. A network is a graphical representation of the molecular relationships between genes or gene products. Genes or gene products were represented as nodes (shapes), and the biological relationship between two nodes was represented as an edge (line). The intensity of the node colour indicates the degree of up-regulation (red) or down-regulation (green). Nodes were displayed using various shapes that represented the functional class of the gene product. Edges were displayed with various labels that described the nature of the relationship between the nodes., which takes into account the calculations of amplification efficiencies and multiple reference genes. Expression levels of target genes were normalised to the geometric mean of the three most stable reference genes, chosen from the four tested, namely RAS p21 protein activator 1 (Rasa1), sema domain Ig domain (Ig) transmembrane domain and short cytoplasmic domain (semaphorin) 4A (Sema4a), protein-tyrosine sulfotransferase 2 (Tpst2), and eukaryotic 18S rRNA endogenous control (18S rRNA). The first three reference genes were selected based on the microarray data, while the fourth is a common reference gene used in real-time qRT-PCR experiments. Stability of these reference genes was assessed using the geNorm 3.5 software .We carried out two-step real-time quantitative RT-PCR (qRT-PCR) studies on target genes selected to represent the tumours used in the microarray experiments, using TaqMan Gene Expression Assays . Primer and probe sets for the selected genes were obtained from the ABI Inventoried Assays-On-Demand (Applied Biosystems). Reactions were carried out according to the manufacturer's instructions. Quality control of the replicates used, real-time qRT-PCR data extraction and initial analysis were carried out using the 7000 Sequence Detection System software (Applied Biosystems). Relative quantification of the target genes of interest was carried out using the qBase 1.3.5 software 25, wht test (comparison of two groups) or ANOVA (comparison of more than two groups) was performed, and significant differences between means were determined. Where appropriate, the required post hoc statistical analyses were applied for a more robust outcome. These are indicated and explained in the appropriate tables and figures. For all outcomes, P< 0·05 was considered statistically significant.Data were analysed using the Statistical Analysis System program. The experimental results are expressed as means and standard deviations, unless otherwise stated. A two-tailed unpaired Student's . The remaining 98 % on a dry weight basis consists of fruit sugars, soluble fibres, organic acids and water-soluble vitamins, etc. It is known that phenolics are minor components in all plants, similar to other phytonutrients such as carotenoids and tocols (vitamin E). However, they are only needed in small quantities to affect a biological response. Since the present study is the first to test the effects of OPP on animals, we arbitrarily chose 500 mg/l GAE as the lowest concentration to investigate whether there were any biological effects on animals. Having determined the positive effects, we moved on to test a whole range of concentrations, with 500 mg/l GAE as the lowest dose and 3000 mg/l GAE as the highest dose. For the anti-diabetic study with Nile rats, a shorter pilot study has indicated that treatment effects could be expected between 900 and 1800 mg/l GAE (data not shown). As such, in the present study, we report the results for the highest dose tested (1800 mg/l GAE). In rat studies, the volume of OPP given was 30–35 ml/d for all concentrations tested. Based on an average rat weight of 300 g, this would be equivalent to the consumption of about 1000 ml of OPP by a 60 kg human subject, which is calculated based on the body surface area normalisation method.OPP in oil palm vegetation liquor ranged from 1·38 to 2·43 % on a dry weight basis10. ThIn vivo animal studies assessed the possible cardioprotective effects of OPP. These include studies on BP regulation in rats treated with l-NAME, an inhibitor of NO synthase, cardiac arrhythmia studies using rat models of coronary artery ligation and atherosclerosis studies using rabbits.l-NAME. At 10 mg/kg, l-NAME resulted in a sharp rise in BP (18 mmHg) in the l-NAME control group  rats developed significantly higher BP values. This protection against hypertension by OPP was greater in rats at the higher dose  for 4 weeks before being treated with ol group . Howeverher dose .Fig. 1Re. In addition to the possible direct effect on the endothelial NO synthase system, OPP being an antioxidant may have scavenged the increased reactive oxygen species associated with l-NAME, thus reducing or inhibiting oxidative stress central to the development of hypertension and atherosclerosis. Therefore, OPP may have broad applications in modulating NO in chronic syndromes such as CVD.Oral administration of OPP thus lowered BP in a NO-deficient rat model of hypertension and is consistent with the observations made with isolated vascular preparations described in the previous study8. In in vivo rat model of cardiac arrhythmia and sudden cardiac death, following a long-term dietary regimen. At the end of the 4-month feeding period, rats were subjected to myocardial ischaemia by ligating and hence occluding the coronary artery. The vulnerability to ventricular tachycardia, VF and sudden cardiac death was measured. The incidence of ventricular tachycardia was not influenced by OPP, but there was a trend (not significant) towards a lower mean duration of ventricular tachycardia  of VF. Of the twenty-five rats, thirteen (52 %) went into VF compared with twenty-one out of twenty-three rats (91 %) in the unsupplemented controls. Rats receiving OPP also had a significantly shortened (P< 0·05 or better) duration of VF. The overall mortality in the OPP group was five out of twenty-five (20 %) compared with nine out of twenty-three (40 %) in the control group. No significant difference was evident in the area of the myocardium that was rendered ischaemic by ligation of the coronary artery, suggesting that these observations were independent of the surgical procedure.The potential cardioprotective effect of OPP was also evaluated using an hycardia . In cont. While the results obtained may not be directly translated to human subjects, it is nonetheless strong evidence of bioactivity and hence bioavailability.As atherosclerosis represents an important component of CVD, the development of atherosclerosis was assessed in rabbits fed a high-fat, cholesterol-rich atherogenic diet. As expected, control rabbits fed this atherogenic diet developed extensive fibrous and fatty plaques as well as fatty streaks in their aortas . On the 28. Whad libitum at 1800 mg/l GAE as the sole drinking fluid for 17 weeks blocked diabetes progression (hyperglycaemia and hyperlipaemia) in 12-week-old male Nile rats as evidenced by normalisation of initially elevated blood glucose and plasma lipids  are good markers of diabetic status and the metabolic syndrome, and all are highly interrelated, as one would predict for diabetes and the metabolic syndrome in human subjects. Elevated TAG, hyperglycaemia, depressed HDL and fatty liver are common to this animal model and the human disease. It is important to note that while some of the seven control rats were diabetic by the end of the present study (blood glucose >1100 mg/l), none of the OPP group was diabetic (blood glucose < 1000 mg/l). The probability that this was not due to chance was significant at P< 0·05, with the means being especially supported by the observation that blood glucose levels in most of the rats receiving OPP declined, whereas those in most of the controls increased. Expressing the data as percentage of change would have been more striking, but that would deprive the reader of appreciating the true biological variation that was observed in the present study. Furthermore, the effect of OPP on diabetes was confirmed statistically (despite the inherent variation) by all the other criteria .Wild-type Nile rats exhibit extensive variation in their rate of developing diabetes17, so and lipid peroxidation, defending pancreatic cells from oxidative damage, protecting lymphocytes from DNA damage, as well as preserving vascular functions in diabetic complications. Mulvihill et al. reported that naringenin attenuated dyslipaemia and hyperinsulinaemia in LDL receptor null mice with diet-induced insulin resistance. Anderson reported that cinnamon polyphenols improved insulin sensitivity in vitro and in individuals with type 2 diabetes. Subjects with the metabolic syndrome who consumed an aqueous extract of cinnamon have been shown to have improved fasting blood glucose, systolic BP, percentage body fat and lean body mass compared with the placebo group. Huang et al. investigated the effects of caffeic and cinnamic acids on glucose uptake by insulin-resistant mouse liver FL83B cells. Their results suggested that these phenolic acids promoted insulin receptor tyrosyl phosphorylation, up-regulated the expression of insulin signal-associated proteins , increased the uptake of glucose, and therefore alleviated insulin resistance in the cells.The present results thus imply that OPP protects against (and may even reverse) early type 2 diabetes characterised by hyperglycaemia and hyperlipaemia in a model that develops spontaneous diabetes when fed typical rat chow. This is in line with the knowledge that phenolics confer anti-diabetic effects via various mechanisms, including reducing oxidative stress30 andInsulin resistance is the main trigger for the metabolic syndrome and is a consequence of the inability of insulin to efficiently signal its receptor kinase and/or downstream targets. Assuming OPP can be found to attenuate metabolic disorders linked to insulin resistance in other systems, it holds promise for this aspect of the metabolic syndrome.ad libitum as the sole fluid source, while the control animals received distilled water. No significant differences were observed in tumour incidence  ncidence , as the ncidence  and weigncidence  in the Ohttp://www.journals.cambridge.org/bjn). On the one hand, genes such as cyclin D2 (Ccnd2), cyclin E2 (Ccne2), cyclin-dependent kinase 4 (Cdk4) and cyclin-dependent kinase 2 (Cdk2) were up-regulated by OPP. On the other hand, genes such as cyclin A2 (Ccna2), cyclin B1 (Ccnb1), polo-like kinase 1 (Plk1), mitotic arrest deficient 2, homologue-like 1 (Mad2l1), cell division cycle 20 homologue (Cdc20), histone deacetylase 6 (Hdac6) and minichromosome maintenance deficient 6 (Mcm6) were down-regulated by OPP. The directions and fold changes for two of these genes, Ccne2 and Ccna2, obtained from real-time qRT-PCR, were comparable with those obtained using the microarray technique , resveratrol, piceatannol, genistein and luteolin. Interestingly, the same genes that we found to be significantly changed by OPP were also found to be regulated, but in the opposite direction, in colorectal tumour cells resistant to a combined chemotherapy of folinic acid, 5-fluorouracil and irinotecan. The opposite direction of regulation of these genes by OPP thus indicates that OPP may help sensitise tumour cells towards chemotherapy, and suggests that it might be used together with selected chemotherapeutic agents for enhanced cytotoxicity in cancer cells.The regulation of cell-cycle genes found in the present study suggests that OPP may inhibit tumour growth ell cycle38. ThStat3) and chemokine (C–X–C motif) ligand 12 (Cxcl12) were also down-regulated by OPP (Stat3 signalling is oncogenic and is required for the transformation of various primary cancer and tumour-derived cell lines. Thus, many cancer-derived cell lines that contain constitutively activated Stat3 are dependent on this protein, and they undergo growth arrest or apoptosis when treated with antisense or dominant negative constructs directed at Stat3. Niu et al. showed that blocking Stat3 in cancer cells up-regulates the expression of p53, leading to p53-mediated tumour cell apoptosis. In addition, inhibition of constitutive Stat3 activity in haematopoietic malignancies such as non-Hodgkin's lymphoma and multiple myeloma sensitises these resistant cancers to chemotherapeutic drug-mediated apoptosis, as well as suppresses the growth of prostate and astrocytoma cancer cells. In agreement with this, resveratrol, which has been reported to have anti-tumour properties, was also found to inhibit Stat3 signalling in transformed mouse fibroblasts as well as human breast, pancreatic and prostate carcinoma cell lines.Furthermore, genes that are normally associated with cancer proliferation and invasion such as signal transducer and activator of transcription 3 (d by OPP . Stat3 s. An important example of a cytokine receptor pair involved in cancer invasion and metastasis is Cxcl12/Cxcr4. In vitro, the CXCL12 ligand stimulated breast cancer cells to carry out the basics of invasion, including pseudopodial protrusion, directed migration and penetration of extracellular matrix barriers. In vivo, metastasis to CXCL12-rich lung tissue was blocked in animal models by treatment with a neutralising anti-human CXCR4 monoclonal antibody. Cxcl12 is also an important mediator of advanced cancer and contributes significantly to the lethal phenotype. Tannic acid, a water-soluble polyphenol, which is abundant in Chinese herbal medicines and tea, is a Cxcl12/Cxcr4 inhibitor, and this activity may contribute to its anti-inflammatory, anti-angiogenic and anti-tumour properties. In the present study, the down-regulation of Cxcl12 in tumours of OPP-treated mice thus suggests a reduction in invasiveness and, possibly, metastatic potential of these tumours.The most invasive cancers normally produce the broadest spectrum and the highest levels of chemokines53. AnThe array of bioactivities displayed by OPP suggests its potential application for a range of chronic diseases. While the biological effects observed in the present study are mainly attributed to phenolic compounds, the possible effects of other components cannot be discounted. What is important is that the extract in its entirety confers the positive outcomes reported in the present study. However, the effects of OPP in human subjects have yet to be shown, and we are currently designing human intervention trials to address this issue. Although we do not have information on the actual bioactive metabolites that reach the target organs, the breadth of biological activities observed in the present study indicates that OPP is bioavailable in animals and triggers a central regulatory response, which modulates key metabolic functions. In addition to the anti-tumour mechanisms observed in the present study, the use of genome-scale techniques to identify the molecular modes of action involved in conferring the biological effects of OPP in other chronic diseases is thus very much called for.