Although β-catenin expression appears to be upregulated and Wnt signaling pathway is activated in the majority of cancers, its expression level seems to be lost in non-small cell lung cancer (NSCLC). We previously reported that the promoter of β-catenin was hypermethylated in two NSCLC cell lines. In the current study, we expanded our analysis for the methylation status of β-catenin promoter region and its protein expression in seven NSCLC cell lines and a series of 143 cases of primary human lung cancer with adjacent non-neoplastic tissues. Quantitative methylation specific PCR (qMSP) analysis showed methylation of β-catenin promoter region in five NSCLC cell lines, with increased β-catenin protein levels upon 5′-Aza-2′-deoxycytidine (5-aza-dC) treatment. The methylation status in SPC (methylated) and A549 (unmethylated) was confirmed by bisulfite sequencing PCR. 5-Aza-dC treatment inhibited invasiveness of SPC but not A549. Immunofluorescence analysis showed membranous β-catenin expression was lost in SPC and could be re-established by 5-aza-dC, while Wnt3a treatment led to nuclear translocation of β-catenin in both SPC and A549. Dual-luciferase assays indicated that 5-aza-dC treatment caused no significant increase in Wnt signaling activity compared with Wnt3a treatment. The effect of demethylation agent in SPC can be reversed by β-catenin depletion but not E-cadherin depletion which indicated that the methylation mediated β-catenin silencing might enhance NSCLC invasion and metastasis in an E-cadherin independent manner. Subsequent immunohistochemistry results further confirmed that β-catenin promoter hypermethylation correlated with loss of immunoreactive protein expression, positive lymph node metastasis, high TNM stage and poor prognosis. The present study implicates β-catenin promoter hypermethylation in the mechanism of epigenetic changes underlying NSCLC metastasis and progression, thus indicating the potential of β-catenin as a novel epigenetic target for the treatment of NSCLC patients. Lung cancer remains a major clinical challenge because of its poor prognosis and limited treatment options Tumor invasion and metastasis occur when tumor cells gain the ability to detach from the primary tumor and enter into surrounding tissue or lymphovascular channels, a process that is critically dependent on the disruption of adhesion junctions between tumor cells et al. showed that the promoter of β-catenin was hypermethylated which led to loss of β-catenin expression in the cell line derived from liver metastasis of gastric cancer but not those primary cancers Aberrant promoter methylation could result in gene silencing in various malignant tumors including lung cancer Here, we studied the impact of the β-catenin promoter methylation statuson tumor progression and invasion, by utilizing both lung cancer cell lines and patients samples as our experimental models.A–IIIB disease.This study was conducted with the approval of the institutional review board at China Medical University . Written consent was given by the participants for their information to be stored in the hospital database for their specimens to be used in this study. All clinical investigation has been conducted according to the principles expressed in the Declaration of Helsinki. One hundred and forty-three cases of primary human lung cancer with corresponding adjacent non-neoplastic tissues who underwent surgical resection with curative intent, were collected from the First Affiliated Hospital of China Medical University between October 2004 and July 2006. Patients' survival was defined as the time from the day of surgery to the end of follow-up or day of death due to recurrence or metastasis. None of the patients received radiotherapy or chemotherapy before surgical resection, and all patients received routine chemotherapy after surgery. The study patient population has been reported in our previous literature Seven NSCLC cell lines, commonly used in our lab, were selected for cell experiments. The HBE, A549 and H1299 cell lines were obtained from the American Type Culture Collection . The PG-BE1 (BE1), PG-LH7 (LH7), SPC-A-1 (SPC), and LTEP-A-2 (LTE) cell lines were obtained from Shanghai Cell Bank . All cells were cultured in RPMI 1640  containing 10% fetal calf serum (Invitrogen), 100 IU/ml penicillin , and 100 µg/ml streptomycin (Sigma). The cells were grown in sterile culture dishes and passaged every 2 days using 0.25% trypsin (Invitrogen).5′-GGAAAGGCGCGTCGAGT-3′ and reverse, 5′-TCCCCTATCCCAAACCCG-3′, with the TaqMan probe 5′-6FAM- CGCGCGTTTCCCGAACCG-TAMRA-3′; β-actin forward, 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′ and reverse, 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′, with the TaqMan probe 5′-6FAM-ACCACCACCCAACACACAATAACAAACACA-TAMRA-3′. The housekeeping β-actin gene (ACTB) was used to normalize a methylation independent control reaction. For relative quantification, amounts of the methylated DNA (percentage of methylated reference) at a β-catenin promoter region were normalized to the methylation value of the calibrator, which was defined as 100%. Universal methylated DNA (QIAGEN) was used as the calibrator. The percentage of methylated reference was defined as 100×2(sampleACTB(ct) − sampleβ-catenin (ct))/2 − calibratorβ-catenin (ct))Genomic DNA was extracted from cell lines and 10-µm-thick sections of 10% neutral formalin-fixed, paraffin-embedded tumor tissue blocks using the QIAamp DNA Mini Kit . Genomic DNA was treated with sodium bisulfite using an EZ DNA methylation kit . Methylation-specific real-time polymerase chain reaction (PCR) assays were performed in the ABI 7900HT Fast Real-time PCR system . Primer pairs were as follows: β-catenin forward, CTNNB1 gene promoter region -1,124–11,114 bp. DNA from lung cancer cells was extracted and then treated with sodium bisulfite using an EZ DNA methylation kit (Zymo Research) according to the manufacturer's instructions. The β-catenin promoter-specific primers for bisulfite sequencing were constructed using promoter sequence data and primer design software ; the primer sequences are shown in We used the Methyl Primer Express v1.0 software  to analyze the Cultivated lung cancer cells were treated with different concentrations of 5-aza-2′-deoxycytidine (5-aza-dC) (Sigma) dissolved in culture medium every 24 hours. Using q-MSP and MTT assays, the drug concentration required for β-catenin promoter CpG island demethylation with no significant effect on cell growth was selected (7 µM) and cells were collected after continued culture for 48 hours .Cells were treated with 500 ng/ml recombinant Wnt3a  for 48 hours.Cell lines were plated in six-well plates with fresh media without antibiotics for 24 h before transfection. Cells were transfected with β-catenin siRNA , E-cadherin siRNA  and control siRNA , using Lipofectamine-2000 (Invitrogen) according to the manufacturer's instruction. All the siRNAs were composed of a pool of two target-specific 19–25 nt siRNAs, The cells were harvested at 48 hours post-transfection.Total protein from cells was extracted in lysis buffer (Pierce) and quantified using the Bradford method. Then, proteins (50 µg) were separated by SDS-PAGE (12%) and transferred to polyvinylidene fluoride (PVDF) membrane . Subsequently, membranes were incubated overnight at 4°C with monoclonal antibodies against E-cadherin , β-catenin  or GAPDH . After incubation with peroxidase coupled anti-mouse IgG (Santa Cruz Biotechnology) at 37°C for 2 h, bound proteins were visualized using ECL (Pierce) and detected using BioImaging Systems . The relative protein levels were calculated based on GAPDH protein as a loading control.5 cells and incubated for 16 hours. Medium supplemented with 10% FBS was added to the lower chamber as the chemoattractant. The numbers of invading cells were counted in 10 randomly selected high-power microscope fields. The experiments were performed in triplicate.Cell invasion assays were performed using a 24-well transwell chamber with a pore size of 8 µm , and the inserts were coated with 20 µl Matrigel . Then, 48 h after transfection, cells were trypsinized and transferred to the upper Matrigel chamber in 100 µl of serum-free medium containing 3×10The cells were fixed with 4% paraformaldehyde, blocked with 1% BSA and then incubated with the β-catenin monoclonal antibody  overnight at 4°C, followed by incubation with fluorescein isothiocyanate (FITC) conjugated secondary antibodies  at 37°C. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). Epifluorescent microscopy was performed using an inverted Nikon TE300 microscope  and confocal microscopy was performed using a Radiance 2000 laser scanning confocal microscope .Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were plated in 24-well plates for 24 h prior to transfection with pGL3-OT or pGL3-OF (0.5 mg) reporter gene plasmids, together with the control plasmid pRL-TK (50 ng). After incubation for 30 h at 37°C, reporter gene expression was detected by the Dual-Luciferase Assay System . Tcf-mediated gene transcription activity was determined by the ratio of pGL3-OT to pGL3-OF luciferase activity, which was normalized to Renilla luciferase activity from the control plasmid pRL-TK. All experiments were performed, in duplicate, a minimum of three times. To analyze the effects of 5-aza-dC and β-catenin siRNA on Wnt signaling, 5-aza-dC was added and β-catenin siRNA was co-transfected with pGL3-OT or pGL3-OF for luciferase assays.Immunohistochemical analysis of β-catenin expression was performed using a β-catenin-specific monoclonal antibody  as previously described and was evaluated according to our previous report P<0.05 was considered to indicate statistically significant differences.SPSS version 13.0 for Windows  was used for all analyses. The chi-square and Spearman's correlation tests were used to examine possible correlations of β-catenin methylation status in NSCLC with its methylation status in adjacent normal lung tissue and clinicopathologic factors. The Mann-Whitney U-test was used to analyze the results of q-MSP, Western blot, Matrigel invasive assays and Dual-luciferase assays. The Kaplan-Meier method was used to estimate the probability of patient survival, and differences in the survival of subgroups of patients were compared using Mantel's log-rank test. Data were presented as the mean of experiments performed in triplicate. The β-catenin promoter methylation levels were tested in lung cancer cell lines. As shown in The chemical 5-aza-dC has been used both in vitro and in vivo to inhibit DNA methylation. In this study, all seven cell lines  were treated with 5-aza-dC . ExpressIn order to further confirm the function of promoter methylation of β-catenin, β-catenin-specific siRNA was introduced into SPC and A549 cells. Compared with cells transfected with control scrambled siRNA, cells transfected with β-catenin siRNA exhibited reduced β-catenin and E-cadherin expression, which cannot be restored by 5-aza-dC treatment . Next, wP<0.001; P = 0.001).We next investigated the methylation status of β-catenin promoter in clinical tissue samples. β-Catenin was expressed predominantly on the cell membrane in normal lung tissue samples . There was no significant association between β-catenin methylation and sex, age, histological type, or differentiation in NSCLC. The Kaplan-Meier test revealed that the overall survival time of NSCLC patients was shorter in patients with β-catenin-methylated tumors than in those with the β-catenin-unmethylated tumors  and MTT (B) were performed. The promoter of β-catenin was demethylated in cells treated with 5-aza-dC in concentration of 7 and 10 µM in both SPC and LTE cells (A). MTT results showed that 7 µM was the minimum concentration for demethylation with relatively small growth inhibition (Bars represent SD. *P<0.05 and ** P<0.01 compared to the 0-µM group).(TIF)Click here for additional data file.Figure S2Effect of 5-aza-dC treatment in the other cell lines with methylations on the β-catenin promoter. Matrigel assay was performed in LH7, BE1, LTE and H1299 cells to test the invading abilities of the cells upon 5-aza-dC treatment  Number of cells invading into the lower surface of the filter was counted, and each experiment was carried out in triplicates. .(TIF)Click here for additional data file.


The theory based on these findings indicates that those who are knowledgeable about their condition, making informed decisions in relation to it, and accept their condition are able to live with it, whilst those who do not accept their condition do not fully adapt to it or integrate it into their lives.A range of literature has explored the experience of living with a long-term condition (LTC), and frequently treats such experiences and conditions as problematic. In contrast, other research has demonstrated that it may be possible to adapt and achieve well-being, even when living with such a condition. This tends to focus on meaning and the qualitative experience of living with an LTC, and offers alternative perspectives, often of the same or similar conditions. As a result of these conflicting views, this study chose to consider two conditions which, though they may lead to life-threatening illness on occasion, do not appear to impact significantly the lives of all those affected on a daily basis. The aim of this research was to explore and explain how people make sense of two long-term, potentially life-threatening health conditions, namely, thrombophilia and asthma. In doing so, it specifically considered the contribution made by information about the condition. A constructivist grounded theory approach was adopted; this enabled the generation of a theory regarding how people make sense of their LTC, whilst acknowledging the social circumstances in which this was situated. Semi-structured interviews were conducted with 16 participants who had given consent to take part in the research. The findings demonstrate that participants undergo a two-stage process— Approximately 20 million people in England have at least a single long-term condition (LTC). The definition of LTC adopted for the purpose of this study is that offered by the Department of Health in England, which states that an LTC is “one that cannot currently be cured but can be controlled with the use of medication and/or other therapies”  and how those with such conditions integrate them into their lives.The aim of this study was to explore and explain how people make sense of two long-term, potentially life-threatening health conditions, namely, thrombophilia, and asthma. It did so by investigating where and how individuals obtained information and its contribution to and usefulness for understanding their condition, by illuminating beliefs and understanding of information about their condition, and examining any other factors which may affect the process experienced by those living with thrombophilia or asthma.A constructivist grounded theory approach was adopted, enabling the production of a theory regarding how people make sense of their LTC. This type of grounded theory contends that knowledge of and meaning in the world is not discovered, but instead constructed and reconstructed by individuals and/or society Charmaz, . ConstruThe perspectives of people with thrombophilia were initially chosen as, unlike many LTCs considered in the literature, this condition did not seem to change people's lives on a day-to-day basis, although it might lead to life-threatening illness on occasion. Following the tenets of grounded theory, theoretical sampling, where the researcher selects individuals for inclusion on the basis of their potential contribution to the further development and explication of the theoretical concepts emerging from the research Charmaz, , was adoAs is usual in qualitative research, an initial literature review was conducted at the beginning of the study in order to identify knowledge gaps and contextualize the research Dunne, . The ideData collection and analysis were conducted iteratively , a national charity which supports those who have had blood clots (thrombophilia only), or a local advert (both thrombophilia and asthma). Recruitment took place between autumn 2008 and spring 2015, by the researcher or colleagues at a local hospital and a national charity. Ethical approval was acquired from the NHS Research Ethics Service (08/H0201/87) and from the university, and permission received from the charity. Participants gave written consent to take part in advance of the interview and also gave verbal consent at the beginning of each interview. Interviews were recorded and transcribed by the researcher, with all identifying features  removed. The main point, however, is that the researcher tried to act ethically throughout the study.The sample involved ten individuals with thrombophilia  and six with asthma . Three participants were formally interviewed a second time.gaining knowledge.A two-stage process was identified which details how people make sense of their LTC, incorporating how people learn about and understand thrombophilia, and, following further theoretical sampling, asthma. For the purposes of this paper, findings are presented in three parts. The findings in relation to those with thrombophilia are detailed first, followed by those relating to asthma, and finally theoretical insights into having an LTC are offered. The first stage is designated The first, pre-diagnosis phase occurs for those individuals who become aware that they may have some sort of illness. This can result from the diagnosis of a family member, a healthcare professional suspecting a particular condition, or from the linking of symptoms by individuals who recognize that these may be related. Some people will experience their condition for some time prior to diagnosis. As a result, individuals will begin to form constructs about their condition.Healthcare professionals had advised several participants that they might have thrombophilia. For example, one man was informed by his healthcare professional that thrombophilia may have been the cause of a deep vein thrombosis (DVT) he had experienced, saying “I'd never heard of it. I'd never heard of Factor V … suggested that I go back to see somebody with an expertise in that sort of thing” (URN P113).It started with my sister, erm she had a problem with her eye and they found out that she had a blood clot behind her eye erm and they did tests for several months because they couldn't work out what it was and then found out that she has the factor V … her doctor at the hospital had told her to tell us to get checked. (P091)Some people had been told by members of their family that thrombophilia existed in the family and that they could be affected. One participant was alerted to the possibility that she may have thrombophilia following her sister's diagnosis.Diagnosis is the point at which individuals received confirmation of their condition, for most people through consultation with a healthcare professional. A number of people were not diagnosed as soon as they experienced symptoms, either due to a missed diagnosis or because the individuals delayed seeking medical advice.I think they diagnosed it pretty early and I think, it didn't get too drastic, sort of erm, a really bad point and they did diagnose it and to be honest with you I do remember having it from, from ever, I always remember having it. (P141)A number of participants with asthma had been diagnosed at a young age and had lived with knowledge of it for most of their lives. They started to understand this when their parents were informed of both diagnosis and ways to manage the condition:These individuals were too young when diagnosed to see this as surprising. It led them to know asthma from a young age and much of the information they gained came from sources other than healthcare professionals.At the time the thrombophilia tests then were really, I mean this is 30 years ago, they were really not very good at all and they at the time it came back and they just looked for things like lupus and stuff like that and it all came back negative so they said no it can't be anything wrong, it's just bad luck. (P111)For those with thrombophilia, in particular, diagnosis was not always a straightforward process. Delayed diagnosis can result from illness prior to the development of appropriate tests, results not being communicated to individuals, or when healthcare professionals do not recognize the cause of symptoms. Those who went undiagnosed experienced further illness before a diagnosis was received and they were provided with appropriate information and advice about the condition. For example, one woman had suffered two clots and received a negative test result.Of those who encountered a delay in diagnosis, most had previously experienced the condition. They were aware of symptoms, had been ill, and, as a result, had gained extensive tentative information before receiving a diagnosis. These experiences thus contributed to individuals’ knowledge, despite often occurring before their condition had been given a name.Subsequent to diagnosis, individuals began to use their knowledge to determine how to deal with and manage the condition on a day-to-day basis so that they experienced well-being. They also amalgamated existing, relevant information with this ongoing knowledge. Information and understanding was usually gained from a number of sources, including healthcare professionals, family and friends, the Internet, books and other media, and, most significantly, experience.My mum's sort of like she had her two penn'orth to add when I was thinking about taking the heparin injection and it's probably the first time she's really said about it to me, apart from the fact she's oh I I don't take ‘em and I've gone through my whole life not knowing you know that I've had it. (P093)One individual had discussed his hereditary thrombophilia with his motherwhilst another had gained information about asthma from friends, saying “There was people at school and stuff that had the asthma, asthma inhalers and, you know, it was, you, you know, I knew quite a lot about it … speaking to some peers and so on” (P145).Experiential knowledge formed a significant aspect of the information held by participants.I don't use my brown inhaler enough because when I'm not ill, say for example over Christmas I had a bit of a cough and a bit of a cold about me so I used my brown inhaler regularly and I was very conscious to take it, but then when I'm feeling fine, I don't really you know, I'll go weeks without taking my blue inhaler and forgetting to take my brown inhaler. And then you know it's only when something triggers it then I'm, oh, quick, have I got the blue inhaler. But then it's silly 'cause I know that taking the brown inhaler regularly really helps, but I do find when I first take it it feels worse … I feel quite tight chested at first. (P141)This included information about illness triggers, symptoms, treatment options, the outcome of not taking treatment as prescribed, the impact of illness on their relationships with healthcare professionals, the long-term effects, and day-to-day experiences of having and managing their condition. This knowledge held more meaning as it was personal to the individual; they found out how the condition affected them personally rather than merely accepting generic information provided by a healthcare professional. One individual said of her asthma inhalers:Not every pain in my leg is is a clot and I've learnt to live with that. Not every pain in my chest is a clot, not every time I have a cough or I feel a bit breathless am I about to die from a pulmonary embolism. (P111)Despite having experienced a pulmonary embolism (PE), one woman with thrombophilia had learned to recognize that day-to-day life with thrombophilia did not necessarily mean she would die of a blood clot.Thus, individuals will gain understanding of their condition from a range of information sources. The Internet can be useful or considered to be too technical, depending on the individual perspective. Personal experiences and the perceived experiences of others were significant sources of knowledge and provided information about what it is like to become ill, what triggers exist to cause illness, and how one can manage the condition on a day-to-day basis.living with a long-term condition. People make informed decisions in relation to their condition. They decide whether or not to seek medical advice when experiencing symptoms, using past experience as well as other information to aid the decision-making process. They choose whether to continue taking medication, and whether to adopt non-drug treatments. In addition, they decide whether to accept risks and consider their lifestyle behaviours.The core category identified in this study is I do sometimes as I said get pains in my legs, but there's only been a couple of times where I've sort of convinced myself that I might have another clot … yeah and I I've never ever since been breathless like I, if I had another PE, boy would I know about it. (P111)Having learnt from previous experiences, those with thrombophilia who had had DVT or PE tended to look for the symptoms experienced when they had thrombosis. For example, one individual who had had both a DVT and a PE looked for the symptoms she had experienced before, leg pain and breathlessness, echoing the stories of other participants.I went off the Coumadin. I haven't had swelling erm, since then, I've increased my running, I haven't broken any bones … it's almost as if right now I'm going back to what I like … it bothers me because I know I'm playing with fire … Do I have a false sense of security because I have been off for six months and this is the longest I've gone without an incident. (P143)Treatment decisions—both taking medical advice and resisting it—were also informed by knowledge. For instance, one woman had made the decision to stop taking warfarin. Despite seven clots, she had experienced severe side effects from the medication. She was fully aware of the risks associated with her decision, but decided that these were outweighed by the benefits at that time.As I've got older, I've found drinking alcohol brings on my asthma. Erm, I don't know why that is, just, and it's only sometimes, only some kinds of alcohol—white wine weirdly seems to bring it on which is odd. (P141)Lifestyle decisions were also made by those with LTCs, often based on experience or knowledge gained in other ways. Those with asthma indicated that alcohol could aggravate their condition, which one woman managed by using her reliever inhaler.Often individuals make decisions informed by knowledge and experience. Some of these are made on a daily basis, for example, taking medication. Others are made only at pertinent times, for example, by those with thrombophilia considering taking a long-haul flight. Individuals also make decisions which, though reversible, are intended to be permanent . These decisions depend on the circumstances at any specific time.By making decisions, individuals take responsibility for managing and a degree of ownership for their condition. A feedback loop exists which enables the outcomes of decisions to contribute to knowledge and thus to influence future choices. This is represented diagrammatically in Some people demonstrated through their comments that they had accepted their condition. This included any uncertainty it created for them and its consequences and implications, including the need to accept responsibility for its management. For those who had experienced a delay in receiving a diagnosis and, particularly, those who had become ill as a result, the past may also need to be accepted. Some individuals do not accept their condition and all its nuances. However, a change in circumstances can cause them to move from acceptance to non-acceptance or vice versa, for example, having recently left military service, one woman had stopped accepting thrombophilia and was questioning both the condition and her approach to its management.Some individuals accepted that they had been advised to take medication to reduce their risk of having a clot, and did so in the knowledge that this was a long-term recommendation and that their medication might have side effects. For example, one individual mentioned that she would prefer not to have to take warfarin—“I'm just waiting for them to invent something else, yeah anything please that won't eat me away inside” (P112).Statistically speaking the chances of my having another clot are only even now about 12% more than the general population … just because you have it doesn't mean to say you're gonna get a clot. It's just one of those horrible things that happens. (P111)Uncertainty featured strongly in the comments of those affected by thrombophilia, with some accepting that the circumstances in which a clot might occur were uncertain, and they could not be sure of the potential cause and timing, whilst others were unable to accept this. One individual, who had experienced a number of clots both personally and of family members, accepted uncertainty as an inherent part of the condition.Similarly, past events were accepted by some participants. Several of those with thrombophilia had inherited the condition, with one saying “At least then I know it's not my fault and anything I've done, it's just the hand I've been dealt I guess” (P112).However, some participants did not accept the uncertainty that results from having a condition such as thrombophilia or previous occurrences. One woman had experienced repeated missed diagnoses over a period of decades and was aware that she may have miscarried as a result of the untreated condition. She struggled to understand how the condition had been missed for so long despite her frequent symptoms and consultations with healthcare professionals—“It's just such a frustrating feeling, you know, and it's not about sort of blame or I'm gonna sue them and all the rest of it, but you just think, god you know, all those flipping opportunities” (P121).Thus, acceptance of the condition, including its implications and consequences, responsibility for managing it, any uncertainty inherent in it, and any adverse events which may have happened, forms a pivotal point for individuals with an LTC. Those who are able to accept their condition, in all its guises, will be more likely to live with it. However, those who do not accept it will live alongside their condition. Despite their lack of acceptance, these individuals had taken some ownership of their condition; they accepted their diagnosis and were making decisions.Living with their condition was possible for some of those interviewed, and this led to it becoming part of their identity. They were aware of it most when it became necessary. People might adopt a changed outlook on life, acquire professional roles relating to their condition, and place it in the context of other illnesses they may have or be aware of.In contrast, other individuals lived alongside their condition, which they found difficult to accept. It caused them concern and restricted their activities whilst drawing their attention. For these people, the condition existed separately from the self and was not part of their identity.Information and knowledge, including that based on experiences, result in constructs which represent a condition as it exists for that individual.Constructs are used as the basis on which individuals make decisions relating to a range of activities. A feedback loop offers further understanding which may reinforce or update existing constructs, or lead to the formation of new ones.Individuals may accept their condition. Acceptance depends on how a person understands and conceptualizes it before diagnosis, at diagnosis, and post-diagnosis in an ongoing process.Individuals who are knowledgeable about their condition, who are able to make informed decisions and who accept the condition may be described as living with the condition. For these people, the condition is part of their identity and integrated into their everyday lives. This may be contrasted with those for whom the LTC exists alongside their selves. For this group, the condition has not become part of the self and their everyday life, but is an unwelcome burden which interrupts their day-to-day life by causing concern and restriction.Based on these findings, it is possible to elucidate a theoretical model of how individuals with an LTC might learn to live with it. This is presented as:This theory is summarized in With regard to how participants obtained information and its usefulness for their understanding of their condition, individuals obtained information from a range of sources. The findings reflect certain aspects of the concept developed by Edwards, Wood, Davies, and Edwards  of ‘distBeliefs about one's condition were an important factor in the decisions individuals made. Choices with regard to medication confirm the findings of previous research, which demonstrates that people consider prescribed medication and choose to take them or not depending on what they consider to be the appropriate option (Atkin & Ahmad, A range of other factors also affected the process experienced by those with the study conditions, including acceptance of any uncertainty caused by the condition, its consequences and implications, and prior events. The concept of acceptance is seen in the literature relating to LTCs and can be linked to mental well-being (McCracken, Concepts similar to that of living with a condition found in the literature include achieving harmony (Delmar et al., This study, using a grounded theory methodology, provides a theoretical model of how individuals with an LTC can make sense of this, learn to adapt to it, and integrate it into their life. Whilst it used the examples of thrombophilia and asthma, the theoretical contribution made can be applied to other conditions which may exist in the background for much of the time, but can on occasion cause life-threatening illness. For many people, an LTC is part of who they are. The condition is recognized as conferring a risk, but is not considered to be especially serious for much of the time. Those affected are able to recognize times when they may be ill and are able to make appropriate decisions to medicate or seek treatment. They are also able to make other decisions, for instance about treatment, often without a great deal of effort because they are aware of the parameters of what they consider to be acceptable. They will return to consider the condition at pertinent times, for example, when needing to make different treatment decisions, but for much of the time it will exist in the background of their lives. Accepting the condition as part of their identity will greatly enhance their well-being.Conversely, for other people, an LTC is a burden. It draws their attention, generates questions, and causes concern. They are unlikely to feel knowledgeable and do not accept all aspects of the condition. In particular, they struggle with the uncertainty inherent in having an LTC whose progression is not necessarily clear, with its consequences or with past events which they may struggle to come to terms with. The condition exists alongside these people and holds a negative meaning; this group is less likely to experience well-being.The theory presented offers an explanation of how those with an LTC can learn to live with it, through the acquisition of knowledge, through making informed decisions, and through acceptance. Awareness of this might be useful for individuals with an LTC, who will be able to use it to identify the circumstances under which they are enabled to live with their condition and thus experience well-being. Health professionals will be able to recognize where they can offer help, advice, and support to aid patients in achieving well-being.


Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells.In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied.Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/β-catenin pathway, observing a loss of nuclear β-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3β and an increase of phosphorylated β-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3β, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3β and β-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed.Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/β-catenin pathway through the increase of Gsk3β expression and β-catenin phosphorylation. Periodontitis is one of the pathologies with highest prevalence  and is aIt is a complex disease caused by multiple stimuli such as inflammation, diabetes, neoplasias, traumas or genetic disorders . ChronicOne of the main stimuli for bone formation is the activation of Wnt/β-catenin pathway. Wnt signaling, which is essential for the osteogenic commitment of progenitor cells such as mesenchymal stem cells has also been shown to be activated during alveolar bone formation ,7. Wnt pOn the other hand Procaine is an anesthetic agent, mainly used for oral surgeries by dentists. It is not cytotoxic and it is approved by the FDA for local skin infiltration and topic use. In addition, procaine inhibits a group of enzymes, DNA-methyltransferases, which are necessary to catalyze DNA methylation ,10. InteTherefore the main aim of this study was to investigate if the administration of procaine to progenitor cells has a negative impact on mineralization and osteo/odontogenesis of progenitor cells by blocking Wnt signaling.All experimental protocols were reviewed and approved by the Ethics Committee for Animal Research of the University of Cordoba and all rats received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research.2 in αMEM + bFGF. Treatments were started, as described below, when cells reached confluence.Euthanasia was performed by aortic puncture and exsanguination under general anesthesia . Tibias and femurs of ten male Wistar rats cut at the epiphyses were perfused with alpha minimal essential medium (αMEM) medium  plus 15% fetal bovine serum (FBS) . Following centrifugation and washing with αMEM medium, bone marrow stem cells were filtered and harvested in 25cm2 flasks  with αMEM + 15% FBS and 1 ng/mL of basic fibroblast growth factor (bFGF) . Fresh αMEM medium with 10% FBS and bFGF was added after 48h and successively changed 2 to 3 times per week. After reaching 90% confluence, cells were collected using Trypsin-EDTA  and harvested in 6-well plates at 13 000 cells/cmOsteo/odontogenesis was induced by treating MSC with 1μM dexamethasone, 10 mM β-glycerolphosphate and 0.2 mM L-ascorbic acid  for 21 days. The effect of procaine during osteo/odontogenesis of MSC or on mature osteo/odontoblasts was studied. For the studies during osteo/odontogenesis, procaine (1 uM)  was administered at the same time that osteo/odontogenic stimulus for 21 days. For the studies on mature osteo/odontoblasts, procaine was administered for 10 days once osteo/odontoblasts were obtained for 21 days with osteo/odontogenic stimulus in MSC. Undifferentiated cells (UC) and osteoblasts like cells (OB) were treated with 10 mM lithium chloride (LiCl) , an activator of the Wnt/β-catenin pathway during 21 days in culture. In addition, MSC were cultured in presence of increasing concentrations of procaine  for 7 days to study the effect of Procaine alone on GSK3β and phospho-β-catenin.Tagln), myocardin (Myocd), runt-related transcription factor 2 (Runx2), osterix , osteocalcin (bone gamma-carboxyglutamate (gla) protein; Bglap), Collagen type I (ColA1), Matrix Gla Protein (MGP), Dentin matrix acidic phosphoprotein (DMP1), Receptor activator of nuclear factor kappa B ligand (RANKL), Sclerostin (SOST), Low density lipoprotein receptor-related protein 5 (Lrp5), Frizzled 1 and Glycogen synthase kinase 3 β (Gsk3β) mRNA levels were determined by quantitative real-time RT-PCR . cDNA was synthesized from 0.5 μg of total RNA with a first strand cDNA synthesis kit  in the presence of random hexamers in a final volume of 20 μl at 25°C for 10 min, followed by 42°C for 15 min and 95°C for 3 min. An RT-PCR SYBR Green kit (Qiagen) was used to quantify mRNA expression levels. All primers, except SOST , were designated with the free Oligo 7 software and their sequences are listed in Total RNA was extracted with TRIzol reagent (Sigma-Aldrich) and quantified by spectrophotometry . Smooth Muscle protein 22 alpha  was stored. Protein concentration was determined by the Bradford assay . For Western Blot, equal amounts of protein were homogenized, followed by electrophoresis in 4–20% SDS-polyacrylamide gradient gel . The protein was transferred to a nitrocellulose membrane , and blots were incubated in blocking solution. Primary antibodies used included goat polyclonal SM22α antibody from Novus Biologicals , (D500), rabbit monoclonal phospho β-catenin , (D1:1000), rabbit monoclonal β-catenin , (D1:1000) and mouse monoclonal GAPDH antibody from Santa Cruz Biotechnology . Blots were immunolabeled using a horseradish peroxidase conjugated secondary antibody (D1:5000) and developed on autoradiographic film using the ECL Western Blotting Detection System from Amersham Biosciences U.K. Limited  in LAS 4000 .™ assay as per manufacturer’s instructions . The kits comprise a 96 well plate on which a consensus sequence for the transcription factor in question has been immobilized . Nuclear extracts are then added to the plates and allowed to bind. Positive controls were also included, namely nuclear extracts from cell lines transfected with constructs over-expressing RunxII. Protein bound to the consensus sequence was then treated with a specific primary antibody, which was then detected using HRP labeled secondary antibody and a colorimetric substrate. Absorbance was measured at 450 nm.RunxII transcription factor activity was determined using a commercial TransAMCells were fixed with cold methanol for 20 min and subsequently washed with PBS. Fixed cells were incubated for 1 h in primary antibodies against β-catenin . Cells were then incubated for 1 h at room temperature with Alexa Fluor 488 anti-mouse secondary antibodies . Cell nuclei were visualized with the nuclear stain 4´, 6-diamino-2-phenylindole dihydrochloride . Pictures were obtained at 40x in Axio Observer.Z1 Inverted Confocal microscope (LSM5 Exciter Zeiss).15 μg of protein were used to colorimetrically (OD 405nm) measure alkaline phosphatase specific activity with 5 mM p-nitrophenolphosphate (Sigma-Aldrich) used as a substrate. Cell lysates were incubated in 2 mM p-nitrophenol phosphate for 30 min at 37°C. The reaction was stopped by adding 1M NaOH, and the product was quantified at 405 nm. One unit of alkaline phosphatase was defined as 1 μmol substrate hydrolyzed per hour (per μg protein/sample).Cells were decalcified with 0.6 N HCl for 24 h, and the calcium content in the HCl supernatant was determined by the o-cresolphthalein complexone method . Cells were washed 3 times with PBS and solubilized in 0.1mol/L NaOH 0.1% sodium dodecyl sulfate (SDS). The calcium content of the cell layer was normalized for total proteinthat was determined by the Bradford assay.Mineral deposition was confirmed by staining with 2% (wt/vol) Alizarin Red S. The cell layers were washed twice with phosphate-buffered saline (PBS), fixed with 2% formaldehyde and 1% sucrose solution for 15 minand then washed with PBS three times. Cells were incubated in 2% alizarin red S solution pH 4.1 with shaking for 20min and subsequently washed five times with pure water pH7 and air-dried. Finally the wells were photographed.2 in αMEM 1 ng/mL of bFGF. The medium was changed 2 to 3 times per week. After reaching 90% confluence, cells were washed with DME without red phenol . 50 μl of MTT (1mg/ml in DME) was added to each well and incubated for another 2 hours. Then, 100 μl of isopropanol  was added to each well until the dark-blue formazan crystals formed dissolved. Finally, the absorbance was measured with a spectrophotometer at 570 nm and normalized at 650 nm. All the tests were carried out in triplicate and compared with control wells in which no procaine was added. Cell survival rate was calculated with the following equation: average absorbance value of experimental group/average absorbance value of control group × 100%.This test was performed at short (48 h) and long (21 days) time culture of cells with different procaine concentrations. The experiments were performed in mesenchymal stem cell (MSC) alone or in (MSC) being differentiated into osteo/odontoblasts. Cell viability was measured at 48h, 7 and 21 days after treatment with increasing concentrations of procaine . The MTT   assay measures the activity of living cells via mitochondrial dehydrogenases. For this, MSC were cultured in 96 multi-well plates 13 000 cells/cmDNA methyltransferase (Dnmt) activity was measured by EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit  using nuclear extracts from undifferentiated (UC), osteo/odontogenic cells (OB) and osteo/odontogenic cells plus procaine (OB+Procaine). Nuclear extracts were isolated with the EpiQuik Nuclear Extraction Kit (Epigentek). Dnmt activity assay was performed according to the manufacturer’s instructions. After incubation, capturing, and developing enzyme activity for samples and controls, absorbance was measured on a microplate reader at 450 nm and Dnmt activity  was calculated according to the following formula: [(sample OD—blank OD)/(sample protein x incubation time)] x 1000.p-value was less than 0.05. Analyses were conducted using SPSS .Data are expressed as mean ± SE. The difference between means from two different groups was evaluated by t test; the difference between means for three or more groups was assessed by ANOVA. Statistical significance was determined according to whether the observed The efficiency of our osteo/odontogenic medium (OB) to obtain odontoblasts has been widely demonstrated . ProcainAt protein level, it was observed that osteo/odontogenesis (OB) increased the content of Runx2 and decreased the amount of SM22α. However the administration of procaine (OB+Proc) led to a contrary effect; decreasing Runx2 transcription factor activity and increasing SM22α protein .The mRNA expression of other genes related to final maturation of odontoblasts such as Dentin Matrix acidic phosphoprotein or RANKL were modified in the same sense; increasing after osteo/odontogenic stimuli (OB) and decreasing with the procaine treatment (OB+Proc), .To evaluate the effects of procaine on mineralization during osteo/odontogenesis of MSC the levels of calcium, alkaline phosphatase activity, Alizarin Red Staining and MGP expression were evaluated. The expression of Wnt/β-catenin related genes such as Frizzled 1, Lrp5/6, GSK3β or SOST was also analyzed. Osteo/odontogenic stimuli (OB) led to the activation of Wnt/β-catenin pathway through the increase of Frizled1 or Lrp5/6  and the During MSC differentiation into osteo/odontoblasts there was a significant down-regulation of GSK3βmRNA. This effect was similar to that observed with LiCl (10 mM) alone or in combination with the stimuli used for osteo/odontoblast differentiation. The addition of procaine plus LiCl to MSC differentiated into osteo/odontoblasts caused elevation of GSK3β expression .In MSC maintained in culture for 7 days, increasing concentrations of procaine  produced a concentration dependent up-regulation of GSK3βm RNA. This acMTT assay was performed to analyze a potential toxic effect of procaine alone  or plus Additional experiments adding procaine for 10 days on mature osteo/odontoblasts were carried out to check the effects of this drug on mature osteoblasts. We observed that procaine administration (OB21 d + (OB+Proc) 10d) induced a decrease of alkaline phosphatase activity  and of tIn order to analyze the demethylant effect of procaine, we measured the global activity of methyltransferases enzymes. Our results showed that during osteo/odontogenic differentiation (OB) there was a significant increase in the activity of methyltransferase enzymes. The treatment with procaine (OB+Proc)at this concentration did not modify the activity of these enzymes with respect to the osteo/odontogenic group .The present study shows that procaine decreases both osteo/odontogenesis and mineralization of bone marrow MSC differentiated into osteo/odontoblasts. This effect is associated with an inhibition of nuclear translocation of β-catenin and Wnt/β-catenin down-regulation. In this study, a demethylant effect of procaine was not observed. In comparison with other studies with this drug, we detect significant differences regarding findings with high doses of procaine (between 1–10 mM). In our work, a final concentration of 1μM of procaine was added and this may be the reason why we did not observe changes in DNA methyltransferase enzymes . HoweverThe action of procaine in the context of mineralization and osteo/odontogenesis is unknown. In planulae and polyps of the hydroid Hydractinia symbiolongicarpus,procaine administration reduced the calcification by calcium carbonate . Data puIn parallel, procaine achieved to decrease both the expression of early markers of osteo/odontogenesis  and mature markers . It is known that during osteo/odontogenic differentiation of bone marrow MSC, β-catenin is translocated into nuclei activating canonical Wnt signaling and promoting or inhibiting the expression of specific genes to achieve osteo/odontoblast differentiation –20. Our With the increase in GSK3β expression, we observed a parallel increase in cytoplasmic phosphorylated β-catenin and a reduction of unphosphorylated β-catenin in the nucleus. These results suggest that procaine reduces differentiation to osteo/odontogenesis by increasing GSK3β and β-catenin phosphorylation with the subsequent potential degradation in the proteasome.Procaine inhibits LiCl induced dephosphorylation promoting GSK3 β expression and Wnt inactivation. This finding is of interest in the interpretation of those pathologies where a canonical Wnt/β-catenin pathway is activated aberrantly, such as some tumoral processes.Our data reveal that the addition of procaine inhibits cell proliferation without producing cell toxicity in undifferentiated MSC or in MSC differentiated into osteo/odontoblasts. This data goes alone with the known effect of inhibition of canonical Wnt signaling by procaine, which decreases cell proliferation.The anti-osteo/odontogenic effects of procaine were also observed on mature cells. This finding illustrates the powerful anti-osteogenic effect of procaine. It is tempting to propose this molecule as an efficient inhibitor of Wnt signaling. However, the required plasmatic concentration to trigger in vivo effects is yet unknown. Others have observed the effects of procaine on Wnt signaling. Gao et al. showed that procaine inhibits Wnt/β-catenin through a promoter demethylation of WIF-1 in lung cancer cells  however Despite the absence of in vivo studies or clinical trials analyzing the role of procaine as a direct inhibitor of canonical Wnt signaling, it would be interesting to test the efficacy of this drug in in vivo studies where nuclear translocation of β-catenin was responsible for the generation of pathological process such as some types of hepatocarcinome or colon cancers.It is unknown if in patients with chronic periodontitis and loss of alveolar bone, an excessive use of procaine might contribute to inhibit Wnt signaling, reducing bone formation and even promoting a loss in the phenotype of cells that compound alveolar bone.In conclusion, our data show a clear anti-osteo/odontogenic effect of procaine on the mineralization and differentiation of rat MSC into osteo/odontoblasts. This effect is mediated through the increase of GSKβ expression and the phosphorylation of cytoplasmic β-catenin inhibiting nuclear translocation of β-catenin and this canonical pathway. These effects at this dose are independent of changes in DNA-methylation.Finally more studies with similar drugs such as lidocaine should be performed to analyze their effect on osteo/odontogenesis and Wnt signaling.S1 FigOsteo/odontogenic stimuli (OB) increased significantly the activity of DNA methyltransferase enzymes. Procaine administration at 1 μM (OB+Proc) does not modify the activity of these enzymes with respect to OB and resulted to be significantly higher than UC (***p<0.001 vs undifferentiated cells (UC).(TIF)Click here for additional data file.


The present study aimed to examine the association between hypoxia-inducible factor (HIF)-1α and the Wnt/β-catenin signaling pathway in a hypoxic environment. The study also aimed to explore the possible mechanisms underlying the invasion of hypoxic gastric cancer cells in vitro and in vivo. The pcDNA™ 6.2-GW/EmGFP-miR-β-catenin plasmid was transfected into SGC-7901 gastric cancer cells, resulting in cells with stable suppression of β-catenin expression. The biological characteristics of the control, liposome, negative control, β-catenin knockdown, hypoxia and hypoxia β-catenin knockdown groups were tested using an invasion assay. The differences in the invasive capacity of the control, negative control and liposome groups were not statistically significant. However, the hypoxia group demonstrated a significantly enhanced invasive capacity, as compared with that in the control group (P<0.05). In the hypoxia β-catenin knockdown group, reduced cell penetration and diminished invasive behavior was observed (P<0.05). In the hypoxia and double  hypoxia groups, HIF-1α, β-catenin, urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP-7) protein and mRNA expression levels were elevated. In response to knockdown of β-catenin expression, HIF-1α, β-catenin, uPA and MMP-7 protein as well as mRNA expression levels were significantly reduced in the hypoxia β-catenin knockdown and the double hypoxia β-catenin knockdown groups. In an in vivo experiment, the growth rate of xenograft tumors of hypoxic and control cells was high alongside increased HIF-1α, β-catenin, uPA and MMP-7 levels according to western blot and immunohistochemical analyses, while growth and protein levels of tumors from hypoxic β-catenin knockdown cells were significantly lower and those of β-catenin knockdown cells were lowest. In conclusion, these results suggested that HIF-1α activation was able to regulate the Wnt/β-catenin pathway, and that HIF-1α may be controlled by the Wnt/β-catenin pathway. A potential mechanism underlying SGC-7901 tumorigenicity is the activation of the Wnt/β-catenin signaling pathway, which activates uPA and MMP-7 expression and contributes to the enhanced invasion of hypoxic cancer cells. The unlimited proliferation of tumor cells can cause hypoxia in tumor tissue. Hypoxia-inducible factor (HIF)-1α is an important transcription factor that regulates oxygen homeostasis, and may be associated with the occurrence of gastric cancer . HIF-1α Invasion and metastasis involve numerous proteins from the matrix metalloproteinase (MMP) enzyme family ,8. MMP-7The present study examined the association between HIF-1α and Wnt/β-catenin in vitro in the SGC-7901 gastric cancer cell line. Furthermore, the signaling involved in metastasis and invasion was determined in vitro and in vivo under hypoxic conditions in SGC-7901 gastric cancer cells. The results of the present study may help to identify molecular targets for improved gastric cancer therapeutic strategies.® 2000 was purchased from Invitrogen Life Technologies. RNAiso Plus, reverse transcription-polymerase chain reaction (RT-PCR) amplification, and reverse transcription kits were all purchased from Takara Biotechnology Co., Ltd. .The SGC-7901 gastric cancer cell line was obtained from the Chongqing Medical University Key Laboratory of Neurology . The RNA interference (RNAi) sequences for pcDNA™6.2-GW/EmGFP-miR-β-catenin (5′-TGAACAAGACGTTGACTTGGA-3′) and the negative control pcDNA™6.2-GW/EmGFP-miR-β-catenin-n (5′-AAATGTACTGCGCGTGGAGAC-3′) were constructed by Invitrogen Life Technologies . RPMI-l640 medium was purchased from HyClone Laboratories, Inc. . Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. . Blasticidin was purchased from Sigma-Aldrich . LipofectamineAnti-GAPDH , horseradish peroxidase (HRP)-conjugated goat anti-immunoglobulin G (IgG), radioimmunoprecipitation lysis buffer, phenylmethyl-sulfonyl fluoride, Bicinchoninic Acid (BCA) Protein Assay kit, Enhanced Chemiluminescence (ECL) reagent, goat anti-rabbit IgG and the immunohistochemistry streptavidin-peroxidase (SP) method chemical kit were purchased from Jiangsu BiYunTian Bio, Ltd. .µmol/l CoCl2   at 37°C in an atmosphere containing 5% CO2 and 25% O2. SGC-7901 cells were cultured under hypoxic conditions in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C, in the presence of 5% CO2, 1% O2 and 94% N2 for 16 h. For double hypoxic conditions, SGC-7901 cells were cultured with 150 MO, USA)  for 8 h µm pore polycarbonate membrane Transwell inserts in a 24-well plate. The control group was cultured under normoxic conditions. The liposome group was cultured under normoxic conditions with the addition of 10 µl liposome culture . The negative control group was cultured under normoxic conditions and transfected with the negative control plasmid pcDNA™6.2-GW/EmGFP-miR-β-catenin-n. The hypoxia group was cultured under physical hypoxic conditions. The hypoxia β-catenin knockdown group consisted of the stably transfected miR-β-catenin-7901 cell line cultured under physical hypoxic conditions.The following five groups were included in the invasion assays: Control, liposome, negative control, hypoxia and hypoxia β-catenin knockdown. The cell migration and invasion assays of the cells were performed as described previously , using 8µl; Life Technologies) was evenly spread on a Boyden chamber membrane (Life Technologies), and placed into 12-well plates. SGC-7901 cells (2×105) were seeded into the wells and cultured in RPMI-1640 supplemented with 5% FBS. The control, liposome and negative control groups were cultured for 24 h under normoxic conditions, whereas the hypoxic and hypoxic β-catenin knockdown groups were cultured for 8 h under normoxic conditions, and then cultured for 16 h under hypoxic conditions. The Boyden chamber was then removed, and the cells that remained on the upper surface were collected using a cotton swab. The cells remaining on the filter were fixed with 4% paraformaldehyde (Life Technologies) for 15 min, dried at room temperature, and stained with hematoxylin and eosin (Life Technologies). Images of the cells that had successfully migrated through the membrane were captured by microscopy  using a Canon camera . At least five different fields  were counted for each experiment, and the results of three independent experiments were averaged.To prepare the artificial basement membrane, Matrigel (40 SGC-7901 cells were divided into the following groups: Control (48 h normoxia), hypoxia , and double hypoxia . In addition, miR-β-catenin-7901 cells were divided into the following groups: Control β-catenin knockdown group, hypoxia β-catenin knockdown group and double hypoxia β-catenin knockdown group, all of which were cultured under the same conditions as listed above. Total RNA was extracted from the cells using RNAiso Plus according to the manufacturer's instructions. Reverse transcription to generate cDNA and PCR amplification were performed using a 7300 Real-Time PCR system . The cycle parameters were set as follows: 94°C for 5 min, then 94°C for 30 sec, 56°C , 55°C (HIF-1α), 57°C (β-catenin), 56°C (uPA) and 57°C (MMP-7) for 35 sec, and 72°C extension for 1 min for 30 cycles; and a final extension at 72°C for 5 min. The primer sequences are shown in µg) were separated by SDS-PAGE (Life Technologies) and transferred to polyvinylidene fluoride membranes. The membranes were primarily blocked with 5% skimmed milk and then incubated with the primary antibodies targeting HIF-1α , β-catenin , uPA , MMP-7  and GAPDH . All antibodies were purchased from Epitomics . The membranes were then incubated with HRP-conjugated secondary antibodies and visualized using the ECL reagent. The film was scanned using a gel imager  and Quantity One 4.62 software was used to analyze the gray values of the protein bands.Total protein was extracted from the six groups of cells, and the protein concentration was measured using the BCA method. Equal amounts of protein  were obtained from the Department of Laboratory Animal Science, Chongqing University Health Science Center . All animals were housed in standard conditions at 26–28°C, with 10 h light/14 h dull light and were given free access to food and water. All of the experiments were performed according to the animal protocol approved by the Institutional Animal Care and Use Committee of Chongqing Medical University . SGC-7901 cells were divided into the following groups: Control (48 h normoxia) and hypoxia . The miR-β-catenin-7901 cells were divided into a control β-catenin knockdown group and a hypoxia β-catenin knockdown group, which were cultured in the same manner as the SGC-7901 groups. The cells from each group were trypsinized, washed in phosphate-buffered saline (Life Technologies) and re-suspended in saline solution (Life Technologies). The nude mice were subcutaneously injected with 5×106 cells per 0.2 ml. Mice were divided into the control, hypoxia, interference and hypoxia interference groups, with five mice in each group. Tumor size was measured every third day. Tumor volume was calculated according to the following formula: V=(axb2)/2, where a was the largest superficial diameter and b the smallest superficial diameter. After four weeks, the mice were sacrificed by cervical dislocation, and the tumors were harvested and images were captured using a Canon IXUS 245 camera (Canon).For the immunohistochemistry experiments, the tumors were harvested, fixed in formalin and embedded in paraffin (Life Technologies), and conventional immunohistochemistry sections were prepared. Detection was performed using the SP method in accordance with standard procedures . PositivFresh tumor tissue from each group was harvested, and total protein was extracted using a protein extraction kit, according to the manufacturer's instructions. Western blots were performed as described above. Blots were quantified using grey scale analysis.Values are expressed as the mean ± standard error of the mean. The significance of differences between the groups was determined using Student's t-test and one-way analysis of variance. Appropriate post hoc tests were used when comparing multiple parameters. All analysis was performed using SPSS 17.0 software . P<0.05 was considered to indicate a statistically significant difference.The invasion assay demonstrated that the number of invaded cells was not significantly different between the control, negative control and liposome groups. However, the number of invaded cells in the hypoxia group was increased as compared with that the control group, while the number in the hypoxia interference group was significantly decreased as compared with that in the hypoxia group .RT-PCR and western blot analyses demonstrated that the mRNA and protein expression levels of β-catenin, HIF-1α, uPA and MMP-7 were low in the SGC-7901 control group, while they were significantly enhanced in the hypoxia group, and in the double hypoxia to an even greater extent (P<0.05)  and 3. TThe mRNA and protein expression levels of β-catenin, HIF-1a, uPA and MMP-7 were increased in the hypoxia β-catenin knockdown group and the double hypoxia β-catenin knockdown group, as compared with those in the control β-catenin knockdown group (P<0.05)  and 3.Compared to the double hypoxia β-catenin knockdown group to the hypoxia β-catenin knockdown group, uPA and MMP-7 mRNA and protein expression levels were significantly increased (P<0.05) .Ten days following inoculation of the cells into nude mice, the cells from the hypoxia group began to form tumors. After 14 days, the cells from the control group also began to form tumors. Finally, after 16 days, the control and hypoxia β-catenin knockdown groups began to form tumors. Following inoculation, time-dependent increases in tumor volume were observed, which were accelerated after 22 days. According to the tumor volume and speed of growth, the tumorigenicity of the hypoxia group was higher as compared with that in the control group and the β-catenin knockdown group , Fig. 4.Four weeks after inoculation, the nude mice were sacrificed. The tumors were embedded in paraffin and immunohistochemistry detected HIF-1α, β-catenin and uPA expression predominantly in the nucleus, while MMP-7 expression was primarily located in the nucleus and the cytoplasm . A lowerHIF-1α, β-catenin, uPA and MMP-7 protein expression levels were highest in the tumor tissue from the hypoxia group and lowest in the interference group. Expression levels of these proteins were significantly reduced in the hypoxia interference group, as compared with those in the hypoxia group (P<0.05) .The carcinogenic activity of the Wnt/β-catenin pathway relies on the accumulation of cytosolic β-catenin. The Wnt signaling pathway is regulated by the levels of β-catenin in the cell; when the levels of β-catenin are elevated, the Wnt pathway is activated . HIF-1α et al (et al (et al (et al (Following suppression of β-catenin expression using RNAi, β-catenin and HIF-1α expression levels were significantly reduced, indicating that HIF-1α levels dropped due to the decrease in β-catenin. These results are concordant with the findings of Kaidi et al  and Lee l (et al . The resl (et al  previousl (et al  suggesteA previous study by our group indicated that hypoxia is able to increase HIF-1α expression, which upregulates MMP-9 and uPA receptor expression in gastric cancer cells, resulting in increased adhesion, migration and invasion . In the According to the in vitro invasion assay, the number of cells transgressing the membrane was increased in the hypoxia group, and invasion of the gastric cancer cells under hypoxic conditions was enhanced. Following knockdown of β-catenin expression, invasiveness was reduced under hypoxia. These results indicated that the absence of oxygen in the environment promotes the expression of HIF-1α, which increases β-catenin expression and activate the Wnt/β-catenin signaling pathway in gastric cancer cells, resulting in increased invasiveness.In the nude mouse xenograft experiment, tumors from the hypoxia group were significantly larger as compared with those from the control group and the hypoxia β-catenin knockdown group, indicating that tumor growth was enhanced for the hypoxia group, as compared with that in the control group. The immunohistochemistry and western blotting results demonstrated that in the xenograft tumors from the hypoxia group, HIF-1α, β-catenin, uPA and MMP-7 protein expression levels were higher as compared with those in the control group and the hypoxia β-catenin knockdown group.In conclusion, the in vitro transwell invasion assay capacity of gastric cancer cells is enhanced under hypoxic conditions when HIF-1α expression is increased by activating the Wnt/β-catenin signaling pathway, subsequently inducing the expression of uPA and MMP-7, which act on the extracellular matrix, promoting cell invasion and the development of gastric cancer.In the present study, RT-PCR and western blot analyses demonstrated that a regulatory interaction exists between the Wnt/β-catenin pathway and HIF-1α. Concurrently, inhibiting β-catenin expression under hypoxic conditions reduced the size of the xenograft tumor as well as uPA and MMP-7 protein expression levels. These results suggested that the ability to enhance gastric cancer cell invasion under hypoxic conditions may be due to an increase in HIF-1α expression, which is real-ized through the activation of Wnt/β-catenin signaling, which can promote uPA and MMP-7 expression. uPA and MMP-7 may then degrade the extracellular matrix in order to promote tumor invasion, thereby contributing to gastric cancer invasiveness.The present study suggested that in gastric cancer cells, HIF-1α has a role both upstream and downstream of the Wnt/β-catenin signaling pathway, and that these pathways have a reciprocal regulative function. Under hypoxic conditions, HIF-1α can facilitate the increased expression of uPA  and MMP-


Our main goal in this review is to discuss the mechanism used by several pathogenic bacteria to modulate the inflammatory response through the Wnt/β-catenin signaling pathway. We think that a deep insight into the role of Wnt/β-catenin signaling in the inflammation may open new venues for biotechnological approaches designed to control bacterial infectious diseases.Innate immunity against pathogenic bacteria is critical to protect host cells from invasion and infection as well as to develop an appropriate adaptive immune response. During bacterial infection, different signaling transduction pathways control the expression of a wide range of genes that orchestrate a number of molecular and cellular events to eliminate the invading microorganisms and regulate inflammation. The inflammatory response must be tightly regulated because uncontrolled inflammation may lead to tissue injury. Among the many signaling pathways activated, the canonical Wnt/ It is also found in adherent junctions, forming a complex with E-cadherin, α-catenin, and actin filaments of the cytoskeleton [β-catenin is an ancient and evolutionary conserved pathway present in lime molds, worms, and humans, with a prominent role in embryogenesis, cell differentiation, and stem cell maintenance self-renewal. Recently, mutations in genes encoding β-catenin or other Wnt pathway components have been identified in certain types of cancer, fibrosis, and inflammatory bowel disease [The protein skeleton . The Wnt disease .β-Catenin is not detectable in the cytoplasm or nucleus in unstimulated cells because free β-catenin levels (the form not bound to the E-cadherin complex) is controlled by the destruction complex. This complex is composed mainly by the tumor suppressor adenomatous polyposis coli (APC) and Axin, which function as scaffolding proteins on which the Ser/Thr kinases casein kinase 1α (CK1α) and glycogen synthase kinase 3β (GSK3β) phosphorylate β-catenin at the N-terminal residues Ser45, Ser33, Ser37, and Thr41 [β-catenin to be polyubiquitinated by the Skp1-Cdc53-F-Box E3 ubiquitin ligase complex (SCFβ-TRCP) and degraded by the proteasome 26S  receptor  proteins. Gene expression ends when the transcription complex is disassembled and nuclear APC exports β-catenin to the cytoplasm where it is degraded [The Wnt/receptor , Wnt On.degraded .β-catenin signaling pathway. A proinflammatory role of Wnt/β-catenin has been recently documented in 3T3-L1 preadipocytes stimulated with Wnt1 [β-catenin pathway with Wnt3a in mouse microglial cells leads to the expression and release of the proinflammatory cytokines interleukins (IL)-6, IL-12, and IFNγ [β-catenin pathway has also been demonstrated in mouse colon epithelial stem cells and macrophages infected with Salmonella [ Mycobacterium [β-catenin pathway downregulates the proinflammatory responses to certain bacterial infections [β-catenin may depend on the stimulus, the cell type, the activation context, and its crosstalk with other signaling pathways. In the next sections, we discuss experimental data demonstrating that Wnt/β-catenin is an important signaling pathway in the regulation of bacterial-induced inflammatory response.Approximately 400 genes involved in cell growth, differentiation, apoptosis, survival, and immune functions are regulated by the Wnt/ith Wnt1 . Activatlmonella  or Mycobacterium , which ifections , 10. Theβ-catenin in Salmonella infections has been investigated in vitro or in streptomycin pretreated mice models [ Salmonella typhimurium causes an increase in GSK3β-dependent β-catenin phosphorylation, leading to an upregulation of IL-6, IL-8, and Wnt2, via TLR5/NF-κB activation [β-catenin mutants that, upon interaction with the NF-κB p50 subunit, decreased the NF-κB DNA binding and transcriptional activities [β-catenin [ Salmonella induced a decrease in Axin1, which is a scaffolding protein controlling the levels of β-catenin [κB proinflammatory pathway by Salmonella is dependent upon β-catenin degradation, suggesting that β-catenin plays a negative regulatory role in the inflammatory response.The role of Wnt/e models , 11–13. tivation , 14. Thetivities . Similar-catenin . These d Salmonella Phopc strain induced an increase of β-catenin phosphorylation at Ser552, a site associated with enhanced transcriptional activity, indicating that a protein effector is responsible for β-catenin stabilization [ Salmonella effector protein AvrA has been shown to affect β-catenin stability because infection of CEC with the Salmonella strain SL1344 Phopc mutant that produces AvrA protein caused β-catenin stabilization, Wnt2/Wnt11 increased expression, and IL-6, IL-4, IFNγ, and TNFα decreased expression [ in vitro and in vivo study has shown that Salmonella SL1344 strain activated NF-κB-dependent Wnt11/Wnt2 and IL-8 expression [βTRCP1-dependent ubiquitination of β-catenin and IκB, the cytoplasmic inhibitor of NF-κB. This AvrA inhibitory activity on ubiquitin ligases would make β-catenin more stable and the NF-κB transcriptional activity less effective  pathogen C. rodentium [β-catenin phosphorylated at Ser552 and the expression of c-myc and cyclin D1. In contrast, IFNγ and TNFα expression in epithelial cells was increased after bacterial infection [IL10−/− mice showed impaired Akt and β-catenin signaling [Stabilization of odentium . Mice trnfection . Interesignaling .β is a downstream effector of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway as well as the Wnt/β-catenin destruction complex that negatively regulates β-catenin. The ability of GSK3β to promote or suppress the inflammatory response depends on the cell type and the stimulus [ C. rodentium infections of CEC, Akt inhibits GSK3β by phosphorylation at Ser9 [β phosphorylation depends on Fzd or TLR activation. Despite this lack of information, it is recognized that activation of the PI3K/Akt and Wnt/β-catenin pathways may cooperate to enhance β-catenin activity in C. rodentium infection mice models , prostaglandin E2 (PGE2), and suppressor of cytokine signaling-3 (SOCS-3), which are involved in downmodulation of the inflammatory response. Interestingly, the effect on genes controlled by Notch1 was dependent on β-catenin and Jagged1, a Notch1 ligand [β-catenin. These data indicate that β-catenin acts at late stages of infection to repress inflammatory programs triggered by TLR2 and iNOS/NO.Macrophages infected with1 ligand . It was  S. typhimurium [ M. tuberculosis, in which Wnt5a and Fzd4 are highly expressed [ M. bovis [κB pathway was responsible for Wnt5a expression and macrophages stimulated with Wnt5a produced IL-12p40 and IFNγ. Given that Wnt5a is able to activate β-catenin dependent and independent signaling, the role of β-catenin was not clearly established in this work. Recently, it was found that activation of the TLR-Myd88-NF-κB pathway in foamy macrophage-like cells obtained from granulomatous lesion of mice infected with Mycobacterium tuberculosis induced the expression of Wnt6 [α expression and triggered macrophage polarization toward an M2 phenotype [ Mycobacterium tuberculosis induces β-catenin stabilization or Wnt5a expression may indicate a pathogenic mechanism whereby bacterial infection is successfully established. Other important and still unresolved issues refer to the Mycobacterium virulence factors responsible for regulating the expression of Wnt ligands and Fzd receptors and whether this regulation takes place in nonprofessional phagocytes.Interestingly Fzd receptors 2, 7, and 8 and Wnt ligands 11 and 2 are upregulated in CEC infected withhimurium  but not xpressed . These dM. bovis . The TLR of Wnt6 . Intereshenotype , 26. Theβ-catenin pathway activation by inhibition of GSK3 activity, promotes host resistance against Pseudomonas aeruginosa infection [ P. aeruginosa express proinflammatory cytokines IL-6, IL1-β, and TNFα, which are directly associated with the severity of the disease in a keratitis mouse model. In this model, β-catenin was degraded in a time-dependent mechanism. Furthermore, overexpression of β-catenin active mutants decreased proinflammatory cytokines, enhanced bacterial clearance, and reduced the severity of the disease caused by this bacterium [β-catenin degradation is required for higher expression of proinflammatory cytokines.Treatment of mouse corneal epithelial cells with LiCl, a compound that mimics Wnt/nfection . Macrophacterium , 26. The P. aeruginosa is able to disrupt the integrity of epithelial cell layer in Caco-2 cells by targeting β-catenin/E-cadherin complex. It was demonstrated that AHL directly interacts with β-catenin, resulting in the hyperphosphorylation of its tyrosine residues and translocation to the cytoplasm where it is phosphorylated by the destruction complex and degraded via the proteasome pathway [ P. aeruginosa strain PAO1, which is a producer of AHL and other quorum sensing molecules, results in high neutrophil recruitment and cytokine production compared with mutant strains unable to secrete this type of molecules [The quorum-sensing molecule acyl-homoserine lactone (AHL) from pathway . Infectiolecules .P. aeruginosa also regulates the c-jun N-terminal kinases (JNKs) activity in corneal epithelial cells [β-catenin at Ser33/37 and Thr41, the same residues that are phosphorylated by GSK3. However, it is not yet clear whether this JNK-dependent β-catenin phosphorylation induces its degradation [ P. aeruginosa express IL-6, IL-8, and TNF-α through a JNK-dependent mechanism [ P. aeruginosa targets β-catenin degradation to induce proinflammatory cytokine expression by secreting AHL and regulating the activity of JNKs.al cells . Interesradation . Finallyechanism . These r Helicobacter pylori resides in the stomach of about half of the world population. The infection with this bacterium is associated with development of gastritis, peptic ulcer, and, in some cases, cancer. Although experimental data have shown that H. pylori induces activation of Wnt/β-catenin signaling through β-catenin stabilization in epithelial cells, a protein effector responsible for β-catenin stabilization has not yet been identified [ H. pylori strains, were not strictly required for β-catenin-induced stabilization [ H. pylori K1 induced phosphorylation of the LRP6 coreceptor at Ser1490 [β-catenin nuclear translocation and transcriptional activity because knockdown of either of these two proteins reduced β-catenin-induced Axin2 expression. Moreover, evidence that LRP6 phosphorylation was dependent on a bacterial effector was based on experiments with H. pylori type four secretion system mutants or heat killed bacteria [β-catenin by H. pylori influences the expression of inflammatory cytokines.The human pathogenentified . The cytlization . Infecti Ser1490 . This phbacteria . Neverthβ-catenin pathway reduces several molecular inflammatory processes that are triggered by bacterial pathogens. The fact that proinflammatory stimuli such as TNFα, IFNγ, and NO are able to increase the expression of Wnt/β-catenin signaling molecules indicate that these pathways should be interconnected. Also, it is likely that proinflammatory stimulation by bacterial infections is a requisite to activate Wnt signaling, indicating that β-catenin activity is required for late stages of the inflammatory response.The inflammatory response is a tightly regulated process because chronic or uncontrolled inflammation may cause tissue injury. Several signaling transduction pathways have been well characterized to induce inflammation; however, much less is known about the suppression and resolution mechanisms that control it. The evidence accumulated so far has pointed out that activation of the Wnt/β-catenin pathway and manipulates cell functions to increase its survival and spread invasion through different mechanisms. Future studies with different pathogenic bacteria and cell types will open new scenarios in which the knowledge of interconnection points in space and time of several signaling pathways may be used to design biotechnological approaches to resolve uncontrolled inflammation and enhance bacterial clearance.It is predictable that different specific combinations of Fzd receptors and Wnt ligands may promote or inhibit inflammation induced by bacterial pathogens. This is because bacterial pathogens modulate distinct key proteins that regulates Wnt


PIK3CA mutations or loss of PTEN expression) and nuclear β-Catenin expression, both variables were determined in 55 matched pairs of primary right sided colon cancer cases with or without distant metastasis. Activating mutations in the PIK3CA gene or loss of PTEN expression did not correlate with distant metastasis while high nuclear β-Catenin expression combined with activation of the PI3K pathway identified cases in which distant metastasis had occurred. Activation of the PI3K pathway was not associated with nuclear β-Catenin expression. We conclude that the transcriptional activity of nuclear β-Catenin depends on PI3K activity. However, PI3K on its own does not affect β-Catenin subcellular localization. Both factors synergize for full WNT signaling activity and are associated with distant metastasis in colon cancer. Thus, the detection of high nuclear β-Catenin expression and simultaneous PI3K pathway activation identifies colon cancer patients with a high risk for distant metastasis.We determined whether active PI3K signaling together with nuclear accumulation of β-Catenin is necessary to fully activate canonical WNT signaling and examined the association of both signaling pathways with colon cancer progression. Using reporter gene assays we examined the activation of β-Catenin mediated transcription upon PI3K inhibition with or without β-Catenin nuclear accumulation. Ectopically induced as well as constitutively active WNT signaling strictly required PI3K activity whereas PI3K inhibition had no effect on β-Catenin subcellular localization but impaired β-Catenin binding to WNT target gene promoters and decreased WNT target gene expression. Transcriptional activity of nuclear β-Catenin depended on active PI3K signaling as nuclear accumulation of β-Catenin failed to induce WNT reporter gene transcription upon PI3K inhibition. PI3K dependend transcriptional transactivation of β-Catenin relies on events beyond phosphorylation at the AKT target site serine 552, as S552D-phosphomimetic β-Catenin mutants were unable to restore WNT signaling when inhibiting PI3K. To study the prognostic value of PI3K pathway activation (activating  Colon cancer (CC) is one of the most prevalent human malignancies, causing more than 50.000 deaths per year in the United States . PrognosAlthough the same APC or β-Catenin mutations are present in all tumor cells of a CC, the WNT pathway is not uniformly active in these tumors. Instead, high WNT activity and nuclear β-Catenin expression often are confined to the tumor margin, reflecting an intratumoral WNT signaling heterogeneity ,7. This PIK3CA gene, encoding the catalytic subunit p110α of PI3K, can be observed in a variety of human malignancies, including colon cancer . Mo. MoPIK3Cn cancer . PI3K aln cancer . Addition cancer . We therAccording to this hypothesis, we here provide mechanistic and clinicopathological evidence that active PI3K signaling is mandatory for full WNT pathway activation, β-Catenin mediated target gene transcription, and tumor progression of CC. Importantly, we demonstrate that the nuclear localization of β-Catenin and its transcriptional transactivation are independent processes, linked by PI3K. Based on these findings, we propose a two-step-model of β-Catenin mediated transcription, in which its nuclear accumulation and subsequent transcriptional transactivation by PI3K are two fundamental steps for WNT signaling activation, driving colon cancer progression.To determine whether PI3K activity is required for β-Catenin transcriptional activation, we used 293T and CHO-1 cells, both of which are devoid of constitutively activating WNT pathway mutations. In these cell lines, we ectopically stimulated WNT signaling using LiCl  and wnt3LY is also known to inhibit Casein kinase 2 (CK2) in vitro , which mTo understand the influence of PI3K signaling on WNT activity and nuclear β-Catenin on a cellular level, we treated cancer cells with LY and monitored WNT activity using TOP-GFP reporters and β-Catenin immunofluorescence. Interestingly, LY treatment significantly reduced GFP fluorescence and thus β-Catenin mediated transcription while, unexpectedly, the subcellular localization of β-Catenin remained unaffected as β-Catenin was found to be expressed equally in the nucleus and the cytoplasm Figure . We confTo investigate if the nuclear import of β-Catenin may happen independently from its transcriptional activation, we fused a myc-tagged modified estrogen receptor ER-myc)  to a degR-myc  toFinally, we confirmed these observations by examining the expression levels of β-Catenin, GFP and phospho-AKT (Ser473) in Western blot analyses. Indeed, following 4-OHT addition, we found a strong induction of β-Catenin mediated transcription as indicated by high GFP expression. This was significantly reduced by inhibiting PI3K signaling with LY. Remarkably, LY treatment even reduced basal β-Catenin mediated transcription, while β-Catenin expression levels were unaffected Figure .Axin2 as shown by decreased enrichment of Axin2 WRE in β-Catenin immunoprecipitated chromatin compared to immunoprecipitated chromatin using control IgG1 , showed no significant association with distant metastasis , routinely screened for mycoplasma contamination and DNA typed to prove the identity of the cell lines used. Cells were cultured in DMEM / 10% FBS , penicillin/streptomycin  at 37°C, 5% CO2 in a humidified incubator and passaged before reaching confluence. The RWP-1 cell line was a kind gift from A.G. de Herreros . The wnt3a-producing L-cells and the corresponding control cells were a kind gift from M. Königshoff . Wnt3a-conditioned medium and corresponding control medium was prepared as previously described . 4-HydroTAM) [TAM  adding a myc tag encoding sequence to its 3′end. Both PCR products were fused in frame using splicing by overlap extension PCR [pcI-neo backbone resulting in pcI-neo-Δ45-β-CateninER-myc. pTOP-Flash, pFOP-Flash and pRenillaTK plasmids were a kind gift of H. Clevers . The pTOP-GFP plasmid resulted from replacing the luciferase gene in pTOP-Flash by the EGFP gene. The plasmids encoding stabilized phosphomimetic S552D-β-Catenin (S552D-Δ45-β-Catenin) or stabilized phosphomimetic S552D-β-Catenin fused to the mutant estrogen receptor (S552D-Δ45-β-CateninER-myc) were constructed using the QuikChange site-directed mutagenesis kit . Correct insertion of the mutations was tested by direct sequencing.The sequence encoding a stabilized deletion mutant β-Catenin (Δ45-β-Catenin) was amplified by PCR from pcI-neo-Δ45-β-Catenin . The sequence encoding the tamoxifen sensitive mutant murine estrogen receptor (ERTAM)  was amplsion PCR  and clon4 cells were seeded in a 24-well plate and transfected with pTOP-Flash or pFOP-Flash respectively, together with pRenillaTK in a 1:10 ratio and the respective plasmid mentioned in the experiment as a total of 250 ng DNA using Fugene 6. After 4 hours, cells were treated as indicated. After 12 hours, dual luciferase reporter assays  were done according to the manufacturer's instructions using an automated Orion II luminometer . TOP / FOP ratios were calculated after normalizing luciferase values to renilla values. All experiments were done in triplicates and repeated at least twice. Representative results are shown.1×103, 0.5 % Na-Deoxycholate, 0.1 % SDS, 1 % Nonidet P-40) containing 1 × PhoStop phosphatase inhibitor cocktail and 1 × Complete proteinase inhibitor cocktail (Roche) according to the manufacturer's instructions. After sonification for 3 seconds at 75% amplitude using an HTU Soni 130 sonifier , debris was removed by centrifugation for 20 min. at 20.000 g and 4°C. Nuclear and cytoplasmic enriched protein fractions were prepared as described previously [Cultured cells were washed within cold PBS, scraped and lysed in triple lysis buffer  and the human acetylcholine receptor promoter (AchR) as control region. The enrichment was expressed as percentage of the input DNA for each condition. To calculate the binding to the DNA region analyzed, the enrichment using the anti-β-Catenin antibody compared to the isotype control IP is expressed as percent of the input as described in detail by Hahn et al [Chromatin immunoprecipitation (ChIP) was performed as previously described . Brieflyhn et al . Primer Cells were seeded at a density of approx. 80% confluence on glass cover slips in a 6 cm diameter cell culture dish. After 12 hours, medium was replaced with fresh medium containing indicated inhibitor or vehicle. After 30 min, cells were transfected with the given plasmids. When needed, the medium was replaced after 4 hours with fresh medium containing 4-OHT or ethanol respectively, together with the inhibitors or vehicle as given. After 24 hours, cover slips were removed, washed in cold PBS, fixed 15 min. with 4% paraformaldehyde / PBS, washed, permeabilized for 10 min. with 0.1 % TritonX-100 and blocked for 1 hour in FBS. The remaining cells in the culture dish were processed for Western blot analysis. Cover slips were incubated with primary antibody diluted 1:100 in 50 % FBS / PBS / 0.05 % Tween-20 for one hour, washed and incubated with Alexa546 or Alexa488 labeled secondary antibody  for 30 min. After washing, cover slips were mounted on a glass slide using Prolong Gold antifade mounting medium (Invitrogen). Images were acquired on a Leica SP5 confocal laser scanning microscope using a 630× oil immersion objective .Archival formalin fixed paraffin embedded (FFPE) tissue of 110 patients that underwent surgical resection of right sided colon cancer (CC) at the University Hospital of Munich was obtained. As a case-control study design, 55 patients without metastatic spread (M0) were matched to 55 cases in which distant metastasis to the liver had occurred (M1), with respect to tumor grade (G), T category and tumor localization. Clinicopathological data was obtained from the Munich Cancer Registry (MKR). The study was performed according to the recommendations of the local ethics committee of the Medical Faculty of the Ludwig-Maximilians Universität München.For the analysis of PTEN expression, the monoclonal mouse anti-human PTEN specific antibody clone 6H2.1  was used at a dilution of 1:30. The specificity and reproducibility of PTEN expression testing using this antibody was previously assayed . For thePIK3CA-mutations were analyzed using pyro-sequencing. Briefly, Hotstar Taq-polymerase  was used together with 1×PCR buffer (1.5 mM MgCl2), 200 µM dNTPs and 400 nM primers applying optimized PCR conditions. PCR products were analyzed using Pyro-Gold kits  together with 3 nM of the corresponding sequencing primer (Table 2) on the Biotage™ Q24 device . Finally, data were analyzed applying the PyroMark™ Q24 software . Primer sequences and analyzed sequences as well as detailed immunohistochemistry protocols can be provided upon request.Tumor tissue to be extracted was marked on H&E stained slides and subsequently extracted from the consecutive unstained dewaxed serial sections using scalpel blades under microscopic control. DNA was isolated from the resulting tissue using DNA-Micro-Amp® Kits  following the manufacturer's instructions. 2-test using SPSS version 20.0 . For all tests a p-value lower than 0.05 was considered as statistically significant.The significance of correlations of the parameters analyzed was calculated applying Pearson's χ