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PDF file - 61KB, Antibodies used to evaluate candidate biomarkers in the TMA.

PDF file - 148KB, Exploratory Baseline Analytes.

Purpose: The EGF receptor (EGFR) and COX2 pathways are upregulated in head and neck squamous cell carcinoma (HNSCC). Preclinical models indicate synergistic antitumor activity from dual blockade. We conducted a randomized, double-blind, placebo-controlled window trial of erlotinib, an EGFR inhibitor; erlotinib plus sulindac, a nonselective COX inhibitor; versus placebo.Experimental Design: Patients with untreated, operable stage II-IVb HNSCC were randomized 5:5:3 to erlotinib, erlotinib–sulindac, or placebo. Tumor specimens were collected before and after seven to 14 days of treatment. The primary endpoint was change in Ki67 proliferation index. We hypothesized an ordering effect in Ki67 reduction: erlotinib–sulindac > erlotinib > placebo. We evaluated tissue microarrays by immunohistochemistry for pharmacodynamic modulation of EGFR and COX2 signaling intermediates.Results: From 2005–2009, 47 patients were randomized for the target 39 evaluable patients. Thirty-four tumor pairs were of sufficient quality to assess biomarker modulation. Ki67 was significantly decreased by erlotinib or erlotinib–sulindac (omnibus comparison, two-sided Kruskal–Wallis, P = 0.04). Wilcoxon pairwise contrasts confirmed greater Ki67 effect in both erlotinib groups (erlotinib–sulindac vs. placebo, P = 0.043; erlotinib vs. placebo, P = 0.027). There was a significant trend in ordering of Ki67 reduction: erlotinib–sulindac > erlotinib > placebo (two-sided exact Jonckheere–Terpstra, P = 0.0185). Low baseline pSrc correlated with greater Ki67 reduction (R2 = 0.312, P = 0.024).Conclusions: Brief treatment with erlotinib significantly decreased proliferation in HNSCC, with additive effect from sulindac. Efficacy studies of dual EGFR–COX inhibition are justified. pSrc is a potential resistance biomarker for anti-EGFR therapy, and warrants investigation as a molecular target. Clin Cancer Res; 20(12); 3289–98. ©2014 AACR.

PDF file - 176KB, Assessing the Adequacy of Randomization.

The hypothesis that the physiologic response to psychologic stress influences the initiation of cancer is highly controversial. The link between initiating stressors, the psychologic stress response, and disease is plausible, considering that the stress response is associated with defined physiologic outcomes and molecular mechanisms. In light of this, we review the clinical relevance of psychologic stress on the risk of cancer, and we propose potential molecular pathways that may link the stress response to early stages of malignant cell transformation.

The response to psychological stress can differ depending on the type and duration of the stressor. Acute stress can facilitate a "fight or flight response" and aid survival, whereas chronic long-term stress with the persistent release of stress hormones such as cortisol has been shown to be detrimental to health. We are now beginning to understand how this stress hormone response impacts important processes such as DNA repair and cell proliferation processes in breast cancer. However, it is not known what epigenetic changes stress hormones induce in breast cancer. Epigenetic mechanisms include modification of DNA and histones within chromatin that may be involved in governing the transcriptional processes in cancer cells in response to changes by endogenous stress hormones. The contribution of endogenous acute or long-term exposure of glucocorticoid stress hormones, and exogenous glucocorticoids to methylation patterns in breast cancer tissues with different aetiologies remains to be evaluated. In vitro and in vivo models were developed to investigate the epigenetic modifications and their contribution to breast cancer progression and aetiology. A panel of triple negative breast cancer cell lines were treated with the glucocorticoid, cortisol which resulted in epigenetic alteration characterised by loss of methylation on promoter regions of tumour suppressor genes including ESR1, and loss of methylation on LINE-1 repetitive element used as a surrogate marker for global methylation. This was verified in vivo in MDA-MB-231 xenografts; the model verified the loss of methylation on ESR1 promoter, and subsequent increase in ESR1 expression in primary tumours in mice subjected to restraint stress. Our study highlights that DNA methylation landscape in breast cancer can be altered in response to stress and glucocorticoid treatment.

Investigational in vitro models that reflect the complexity of the interaction between the immune system and tumours are limited and difficult to establish. Herein, we present a platform to study the tumour-immune interaction using a co-culture between cancer spheroids and activated immune cells. An algorithm was developed for analysis of confocal images of the co-culture to evaluate the following quantitatively; immune cell infiltration, spheroid roundness and spheroid growth. As a proof of concept, the effect of the glucocorticoid stress hormone, cortisol was tested on 66CL4 co-culture model. Results were comparable to 66CL4 syngeneic in vivo mouse model undergoing psychological stress. Furthermore, administration of glucocorticoid receptor antagonists demonstrated the use of this model to determine the effect of treatments on the immune-tumour interplay. In conclusion, we provide a method of quantifying the interaction between the immune system and cancer, which can become a screening tool in immunotherapy design., Al-Hity et al present a platform to study the tumour-immune interaction using a co-culture between cancer spheroids and activated immune cells. They assess the effects of cortisol application to the culture model as proof-of-concept, which demonstrates the potential for this platform as a screening tool for immunotherapy design.

People that develop extracranial cancers often display co-morbid neurological disorders, such as anxiety, depression and cognitive impairment, even before commencement of chemotherapy. This suggests bidirectional crosstalk between non-CNS tumours and the brain, which can regulate peripheral tumour growth. However, the reciprocal neurological effects of tumour progression on brain homeostasis are not well understood. Here, we review brain regions involved in regulating peripheral tumour development and how they, in turn, are adversely affected by advancing tumour burden. Tumour-induced activation of the immune system, blood-brain barrier breakdown and chronic neuroinflammation can lead to circadian rhythm dysfunction, sleep disturbances, aberrant glucocorticoid production, decreased hippocampal neurogenesis and dysregulation of neural network activity, resulting in depression and memory impairments. Given that cancer-related cognitive impairment diminishes patient quality of life, reduces adherence to chemotherapy and worsens cancer prognosis, it is essential that more research is focused at understanding how peripheral tumours affect brain homeostasis.

Introduction The aim of the study was to evaluate the reliability of five different sweat analysis techniques which measure; whole body sweat rate [WBSR], local sweat rate [LSR] (via technical absorbent [TA] method and KuduSmart® monitor), sweat conductivity [SC] and sweat gland activation [SGA] in a female population when exercising moderately under heat stress. Methods Fourteen females (age; 26 ± 7 years, body mass; 66.5 ± 7.6 kg, height; 167.1 ± 6.4 cm) completed a preliminary threshold walking test (to determine exercise intensity) and two main trials, separated by 2 days. Main trials consisted of 30-min seated rest in the environmental chamber (35 °C, 50% relative humidity) in an upper body sauna-suit, before its removal, and walking at a moderate intensity (4 metabolic equivalents) for 30-min (speeds ranged from 4.8 to 6.5 km h−1). WBSR was measured via nude mass pre and post exercise. The TA and Tegaderm patches (for sweat sodium chloride) were placed on the back, forearm and chest for the entire 60-min, replicated for all participants for both trials. SGA was assessed following the 60-min trial and the KuduSmart® monitor was placed on the left arm for the 30-min of exercise. Results WBSR, LSR methods and SC demonstrated no difference between trials (p > 0.05), good agreement (within limits), strong correlations (r ≥ 0.88) and low typical error of measurements [TEM] ( Conclusion Sudomotor function is reliable, as demonstrated by good reliability, small TEM and strong correlations. The use of these sweat techniques is appropriate and practical in females who are exercising at moderate intensity under heat stress, and so, may aid future interventions. SGA shows larger variation and should be used with caution.

Hydrogen peroxide (H2O2) is a molecule that plays an important role in cancer. Low concentrations in H2O2 have an important physiological role in signalling whilst higher concentrations can induce cell death. Current sensors for cellular monitoring of H2O2 are fragile and widely used for recording over a duration of minutes. Our study focused on developing a robust sensing device that can be used for sustained cellular monitoring of H2O2 to provide vital insight into its role in cancer. We made composite electrodes using varying compositions of multiwall carbon nanotubes (MWCNTs) and platinum black (PtB). These electrodes were investigated using different electroanalytical approaches, scanning electron microscopy and energy dispersive X-ray spectroscopy. We found that a composition containing 15% MWCNT and 20% PtB was the most sensitive for H2O2 detection. This electrode had a limit of detection of 17 nM and a sensitivity of 73.3 ± 1.1 A M−1 cm−2 when normalised for the conductive surface area. The composite electrode was able to provide stable current responses over 200 min. To evaluate the sensor, measurements in breast cancer cells were conducted, where administration of the pro-oxidant tert-Butyl hydroperoxide increased H2O2 levels. This response was validated using a reactive oxygen species sensitive dye and confocal imaging. Our findings showcase that MWCNT PtB composite electrodes have excellent sensitivity and provide the ability to conduct sustained measurement. This approach towards sensor development can be suitable for monitoring different reactive species which are of paramount importance in complex biological environments.

Social isolation (SI) is a major health risk in older people leading to cognitive decline. This study examined how SI and age influence performance in the novel object recognition (NOR) and elevated plus maze (EPM) tasks in C57BL/6 mice aged 3 or 24 months. Mice were group-housed (groups of 2-3) or isolated for 2 weeks prior to experimentation. Following NOR and EPM testing hippocampal norepinephrine (NE), 5, hydroxytryptamine (5-HT), 5, hydroxyindole acetic acid (5-HIAA), corticosterone (CORT) and interleukin-6 (IL-6) were determined and serum collected for basal CORT analysis. A separate set of mice were exposed to the forced swim test (FST), sacrificed immediately and serum CORT determined. SI impaired performance in the NOR and the FST, reduced hippocampal 5-HT, increased hippocampal IL-6 and increased serum CORT post-FST in young mice. Aged mice either failed to respond significantly to SI (NOR, FST, hippocampal 5-HT, serum CORT post FST) or SI had synergistic effects with age (hippocampal NE, 5-HIAA:5-HT). In conclusion, the lack of response to SI in the aged mice may affect health by preventing them adapting to new stressors, while the synergistic effects of SI with age would increase allostatic load and enhance the deleterious effects of the ageing process.

the aim of this study was to assess the validity of a novel wearable sweat rate monitor against an array of sweat analysis techniques which determine sudomotor function when exercising moderately under heat stress. Construct validity was determined utilising a 5-day short-term heat acclimation (STHA) intervention.Nineteen healthy individuals (age: 41 ± 23 years, body mass: 74.0 ± 12.2 kg, height: 174.9 ± 6.9 cm) [male; n = 15, female; n = 4] completed nine trials over a three-week period, in a controlled chamber set to 35 °C, 50% relative humidity for all sessions. The pre and post-trials were separated by five consecutive controlled hyperthermia HA sessions. Sweat analysis was compared from pre and post-trial, whereby whole body sweat rate (WBSR) was assessed via pre and post nude body mass. Local sweat rate (LSR) was determined via technical absorbent patches (TA) (weighed pre and post) and a novel wearable KuduSmart® (SMART) monitor which was placed on the left arm during the 30-min of exercise. Tegaderm patches, used to measure sweat sodium chloride conductivity (SC), and TA patches were placed on the back, chest and forearm for the 30-min cycling.Sudomotor function significantly adapted via STHA (p 0.05); demonstrated by a WBSR increase of 24%, LSR increase via the TA method (back: 26%, chest: 45% and arm: 48%) and LSR increase by the SMART monitor (35%). Finally, SC decreased (back: -21%, chest: -25% and arm: -24%, p 0.05).All sweat techniques were sensitive to sudomotor function adaptation following STHA, reinforcing their validity. The real time data given by the wearable KuduSmart® monitor provides coaches and athletes instant comparable sudomotor function feedback to traditional routinely used sweat analysis techniques.

Patients diagnosed with cancer often undergo considerable psychological distress, and the induction of the psychological stress response has been linked with a poor response to chemotherapy. The psychological stress response is mediated by fluctuations of the hormones glucocorticoids (GCs) and catecholamines. Binding to their respective receptors, GCs and the catecholamines adrenaline/noradrenaline are responsible for signalling a wide range of processes involved in cell survival, cell cycle and immune function. Synthetic GCs are also often prescribed as co-medication alongside chemotherapy, and increasing evidence suggests that GCs may induce chemoresistance in multiple cancer types. In this review, we bring together evidence linking psychological stress hormone signalling with resistance to chemo- and immune therapies, as well as mechanistic evidence regarding the effects of exogenous stress hormones on the efficacy of chemotherapies.

Minimally invasive, reliable and low-cost in vivo biosensors that enable real-time detection and monitoring of clinically relevant molecules and biomarkers can significantly improve patient health care. Microneedle array (MNA)-based electrochemical sensors offer exciting prospects in this respect, as they can sample directly from the skin. However, their acceptability is dependent on developing a highly scalable and cost-effective fabrication strategy. In this work, we evaluated the potential for poly(lactic acid)/carboxyl-multiwalled carbon nanotube (PLA/ f-MWCNT) composites to be developed into MNAs and their effectiveness for dermal biosensing. Our results show that MNAs are easily made from solvent-cast nanocomposite films by micromolding. A maximum carbon nanotube (CNT) loading of 6 wt % was attained with the current fabrication method. The MNAs were mechanically robust, being able to withstand axial forces up to 4 times higher than necessary for skin insertion. Electrochemical characterization of these MNAs by differential pulse voltammetry (DPV) produced a linear current response toward ascorbic acid, with a limit of detection of 180 μM. In situ electrochemical performance was assessed by DPV measurements in ex vivo porcine skin. This showed active changes characterized by two oxidative peaks at 0.23 and 0.69 V, as a result of the diffusion of phosphate-buffered saline. The diagnostic potential of this waveform was further evaluated through a burn wound model. This showed an attenuated oxidative response at 0.69 V. Importantly, the impact of the burn could be measured at progressive distances from the burn site. Overall, alongside the scalable fabrication strategy, the DPV results promise efficient electrochemical biosensors based on CNT nanocomposite MNAs.

Stress hormones have been shown to be important mediators in driving malignant growth and reducing treatment efficacy in breast cancer. Glucocorticoids can induce DNA damage through an inducible nitric oxide synthase (iNOS) mediated pathway to increase levels of nitric oxide (NO). Using an immune competent mouse breast cancer model and 66CL4 breast cancer cells we identified a novel role of NOS inhibition to reduce stress-induced breast cancer metastasis. On a mechanistic level we show that the glucocorticoid cortisol induces expression of keys genes associated with angiogenesis, as well as pro-tumourigenic immunomodulation. Transcriptomics analysis confirmed that in the lungs of tumour-bearing mice, stress significantly enriched pathways associated with tumourigenesis, some of which could be regulated with NOS inhibition. These results demonstrate the detrimental involvement of NOS in stress hormone signalling, and the potential future benefits of NOS inhibition in highly stressed patients.

Background Epigenetic changes are highly responsive to environmental changes, including stress. The release of stress hormones; such as glucocorticoids, in response to stress have been shown to induce epigenetic modifications in neuronal cells. However, the role of glucocorticoids on epigenetic changes underlying important processes such as cell cycle regulation, apoptosis, and proliferation in breast cancer are not yet established. Furthermore, it is not known if cortisol can induce irreversible epigenetic changes on these cellular processes and whether these changes relate to the duration of the stress response. In this study, cortisol-induced epigenetic changes and the potential involvement of DNA methylation was assessed. Methods We analyzed the expression levels of maintenance DNA methyltrasferase (DNMT1) in MDA-MB-231, Hs-578T, MCF7, and T47D breast cancer cell lines by real-time PCR. We also used Qiagen Epitect Methyl ll Complete PCR array for Tumour Suppressor genes to analyse the level of methylation in 94 tumour suppressor genes. The methylation level on the Long Interspersed Nuclear Element (LINE-1) was used as surrogate marker for global DNA methylation. Results Our results show that cortisol significantly decreased the expression of DNMT1 in the triple negative cells lines MDA-MB-231 (p Conclusions Taken together, cortisol induced aberrant methylation patterns which may have important implications on progression of the disease. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background: The mechanisms by which stress hormones impact triple-negative breast cancer (TNBC) etiology and treatment are unclear. We have previously shown that stress hormones, cortisol, and catecholamines induce rapid DNA damage and impact DNA repair in NIH 3T3 fibroblasts. This study investigates whether stress hormones increase DNA damage in breast cancer cells and if this impacts drug efficacy. Methods: We first screened a panel of 39 breast cancer cell lines for expression of adrenergic and glucocorticoid receptors and examined if stress hormones induce DNA damage and alter cell cycle regulation in vitro. A TNBC xenograft model was used to assess the impact of restraint stress on tumour growth and chemosensitivity to paclitaxel. Results: We found that stress hormones induced DNA damage, phosphorylation of ATR, which was accompanied by an up-regulation of the G1 cell kinase inhibitor p21 and a cell cycle halt of TNBCs in the G1 phase. p21 knockdown abrogated G1 arrest by stress hormones. We also demonstrated that stress significantly decreased efficacy of paclitaxel. Conclusion: We describe a novel mechanism through which stress hormones can induce drug resistance to paclitaxel, which may have profound implications for treating drug resistance in patients with TNBC. Fil: Reeder, A.. University of Pittsburgh; Estados Unidos Fil: Attar, M.. University of Pittsburgh; Estados Unidos Fil: Nazario, L.. University of Pittsburgh; Estados Unidos Fil: Bathula, C.. University of Pittsburgh; Estados Unidos Fil: Zhang, A.. University of Pittsburgh; Estados Unidos Fil: Hochbaum, Daniel. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Roy, E.. University of Pittsburgh; Estados Unidos Fil: Cooper, K. L.. University of Pittsburgh; Estados Unidos Fil: Oesterreich, S.. University of Pittsburgh; Estados Unidos Fil: Davidson, N. E.. University of Pittsburgh; Estados Unidos Fil: Neumann, C. A.. University of Pittsburgh; Estados Unidos Fil: Flint, M. S.. University of Pittsburgh; Estados Unidos. University of Brighton; Reino Unido

The ability of stress to induce immune suppression is widely recognized, but the mechanisms underlying the effects of stress on the adaptive immune system during tumor progression are not completely understood. To study the effect of stress on the immune system in vivo, we used a preclinical immunocompetent mouse model bearing 4T1 mammary adenocarcinoma cells. Mice were randomized into 4 groups, including social isolation (SI), acute restraint stress (aRRS), chronic restraint stress (cRRS), or no stress (NS). We found that SI significantly decreased the number of tumor-bearing mice still alive at the end of protocol (28 days), compared to NS mice. Although we did not detect significant changes in primary tumor volume, we observed a significant increase in the endothelial marker CD31 in primary tumors of SI mice and in lung metastases in SI and RRS mice. Survival decline in SI mice was associated with significant decreases in splenic CD8 cells and in activated T cells. From a mechanistic standpoint, RRS increased expression of FOXP3, CXCL-10, and granzyme B in mouse tumors, and the effects were reversed by propranolol. Our data demonstrate that various forms of stress differentially impact adaptive immunity and tumor angiogenesis, and negatively impact survival.

BACKGROUND Esophageal adenocarcinoma (EAC) is associated with a dismal prognosis. The identification of cancer biomarkers can advance the possibility for early detection and better monitoring of tumor progression and/or response to therapy. The authors present results from the development of a serum-based, 4-protein (biglycan, myeloperoxidase, annexin-A6, and protein S100-A9) biomarker panel for EAC. METHODS A vertically integrated, proteomics-based biomarker discovery approach was used to identify candidate serum biomarkers for the detection of EAC. Liquid chromatography-tandem mass spectrometry analysis was performed on formalin-fixed, paraffin-embedded tissue samples that were collected from across the Barrett esophagus (BE)-EAC disease spectrum. The mass spectrometry-based spectral count data were used to guide the selection of candidate serum biomarkers. Then, the serum enzyme-linked immunosorbent assay data were validated in an independent cohort and were used to develop a multiparametric risk-assessment model to predict the presence of disease. RESULTS With a minimum threshold of 10 spectral counts, 351 proteins were identified as differentially abundant along the spectrum of Barrett esophagus, high-grade dysplasia, and EAC (P

Purpose: The EGF receptor (EGFR) and COX2 pathways are upregulated in head and neck squamous cell carcinoma (HNSCC). Preclinical models indicate synergistic antitumor activity from dual blockade. We conducted a randomized, double-blind, placebo-controlled window trial of erlotinib, an EGFR inhibitor; erlotinib plus sulindac, a nonselective COX inhibitor; versus placebo. Experimental Design: Patients with untreated, operable stage II-IVb HNSCC were randomized 5:5:3 to erlotinib, erlotinib–sulindac, or placebo. Tumor specimens were collected before and after seven to 14 days of treatment. The primary endpoint was change in Ki67 proliferation index. We hypothesized an ordering effect in Ki67 reduction: erlotinib–sulindac > erlotinib > placebo. We evaluated tissue microarrays by immunohistochemistry for pharmacodynamic modulation of EGFR and COX2 signaling intermediates. Results: From 2005–2009, 47 patients were randomized for the target 39 evaluable patients. Thirty-four tumor pairs were of sufficient quality to assess biomarker modulation. Ki67 was significantly decreased by erlotinib or erlotinib–sulindac (omnibus comparison, two-sided Kruskal–Wallis, P = 0.04). Wilcoxon pairwise contrasts confirmed greater Ki67 effect in both erlotinib groups (erlotinib–sulindac vs. placebo, P = 0.043; erlotinib vs. placebo, P = 0.027). There was a significant trend in ordering of Ki67 reduction: erlotinib–sulindac > erlotinib > placebo (two-sided exact Jonckheere–Terpstra, P = 0.0185). Low baseline pSrc correlated with greater Ki67 reduction (R2 = 0.312, P = 0.024). Conclusions: Brief treatment with erlotinib significantly decreased proliferation in HNSCC, with additive effect from sulindac. Efficacy studies of dual EGFR–COX inhibition are justified. pSrc is a potential resistance biomarker for anti-EGFR therapy, and warrants investigation as a molecular target. Clin Cancer Res; 20(12); 3289–98. ©2014 AACR.

Treatment for chronic constipation in older people is challenging and the condition has a major impact on quality of life. A lack of understanding about the causes of this condition has hampered the development of effective treatments. 5-HT is an important pro-kinetic agent in the colon. We examined whether alterations in colonic 5-HT signalling underlie age–related changes in faecal output in mice and whether these changes were due to an increase in TNF-α. Components of the 5-HT signalling system (5-HT, 5-HIAA, SERT) and TNF-α expression were examined in the distal colon of 3, 12, 18 and 24-month old mice and faecal output and water content monitored under control conditions and following the administration of etanercept (TNF-α inhibitor; 1 mg Kg−1). Faecal output and water content were reduced in aged animals. Age increased mucosal 5-HT availability and TNF-α expression and decreased mucosal SERT expression and 5-HIAA. Etanercept treatment of old mice reversed these changes, suggesting that age-related changes in TNFα expression are an important regulator of mucosal 5-HT signalling and pellet output and water content in old mice. These data point to “anti-TNFα” drugs as potential treatments for age-related chronic constipation.

Epinephrine and norepinephrine are produced during psychological stress and can directly bind to cells to induce DNA damage. These effects may have more long-lasting consequences such as DNA mutations resulting in an increased potential for cellular transformation and/or tumor progression. This study examined the molecular effects of a chronic (24 h) in vitro exposure to these stress hormones on murine 3T3 cells. Long exposures (24 h) in dose-response experiments with norepinephrine or epinephrine induced significant increases in DNA damage in treated cells compared to that of untreated controls as measured by the alkaline comet assay. Pre-treatment with a blocking agent (the β-adrenergic receptor antagonist propranolol) eliminated this increase in damage. In addition, both norepinephrine and epinephrine increased cellular transformation, as assessed by growth in soft agar, and 3T3 cells pre-treated with either norepinephrine or epinephrine induced a more rapid onset of tumors and more aggressive tumor growth in nude mice. In summary, incubation of 3T3 cells with catecholamines results in long-term DNA damage as measured by increased transformed phenotypes and tumor progression, indicating that they are important mediators of stress effects on genomic instability and vulnerability to tumor formation.

OBJECTIVE: The present study aimed to identify differentially expressed proteins employing a high resolution mass spectrometry (MS)-based proteomic analysis of endometrial cancer cells harvested using laser microdissection. METHODS: A differential MS-based proteomic analysis was conducted from discrete epithelial cell populations gathered by laser microdissection from 91 pathologically reviewed stage I endometrial cancer tissue samples (79 endometrioid and 12 serous) and 10 samples of normal endometrium from postmenopausal women. Hierarchical cluster analysis of protein abundance levels derived from a spectral count analysis revealed a number of proteins whose expression levels were common as well as unique to both histologic types. An independent set of endometrial cancer specimens from 394 patients were used to externally validate the differential expression of select proteins. RESULTS: 209 differentially expressed proteins were identified in a comparison of stage I endoimetrial cancers and normal post-menopausal endometrium controls (Q

Psychological stress is recognized as a factor that contributes to increased susceptibility to a number of diseases, including cancer. Psychological stress, via release of chemical mediators, can induce long-term changes in the organism resulting in an altered responsiveness of the organism to external carcinogens. The present investigation sought to evaluate the impact of stress on polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. We utilized a repetitive restraint stress mouse model and the model PAH, 7,12-dimethylbenz[a]anthracene (DMBA). Restraint stress was applied three times a week for 6 weeks and DMBA was administered intra-gastrically once a week for 6 weeks and formation of skin, mammary and ovarian tumours was examined at 26 weeks by pathological analyses. The results indicate that stress accelerates the onset of tumour formation produced in response to DMBA and significantly influences the type and location of tumours. Taken together, these results demonstrate that stress enhances the carcinogenicity of DMBA. These results clearly indicate the need for further characterization of the impact of stress on carcinogen metabolism in oncology. Copyright © 2010 John Wiley & Sons, Ltd.

Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma, and understanding how the local microenvironment at the melanoma site influences this progression, are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs), and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests analyzed by nanoflow liquid chromatography-tandem mass spectrometry with an LTQ-Orbitrap. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, non-involved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment.

Polycyclic aromatic hydrocarbons (PAHs) are environmental carcinogens implicated to underlie development of several types of cancers. Cytochrome P450 (CYP) enzymes play key roles in the conversion of PAHs to highly potent carcinogens, namely diol epoxides. 7,12-Dimethylbenz(a)anthracene (DMBA), a PAH, is highly carcinogenic, where in mouse models it is known to be responsible for initiating tumor formation in many organs including mammary tissues, ovaries, and skin. Psychological stress, via release of biochemical mediators, can greatly impact carcinogenesis. The present investigation examined the hypothesis that psychological stress modulates metabolism and carcinogenicity of DMBA through alteration of key drug metabolizing enzyme abundance levels in the liver utilizing mass spectrometry-based proteomics. To test this hypothesis, four groups of mice were treated as follows: nonstressed, stressed, nonstressed/DMBA-exposed, and stressed/DMBA-exposed, where the stressor was a well-accepted model of restraint. Liver proteins were extracted, resolved by one-dimensional gel electrophoresis, digested in-gel with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. This investigation resulted in the unique identification of 59 isoforms of CYP enzymes. Changes in protein abundances derived from spectral counting indicates that stress alone results in increases in the abundance of proteins responsive to oxidative stress, along with Phase I and II metabolizing enzymes, such as CYP2J5 and UDP glucoronytransferases. The proteomic results further indicate that exposure to DMBA induces increases in the abundance of CYP1A2 and serine protease inhibitors and decreases the abundance of CYP4 V3. Finally, significant changes in the abundance of proteins such as CYP1A2, CYP3A11, and Topoisomerase-2 were found between nonstressed and stressed/DMBA-treated mice. These data support the hypothesis that psychological stress modulates DMBA-induced regulation of drug metabolizing proteins in the liver.

Habitual aerobic exercise has a beneficial effect on many systems of the body, and psychological stress has a negative influence on several of the same systems. One possible pathway is through those systems that account for the detrimental effects of stress; by buffering these harmful effects, exercise may reduce the consequences of stress. This study examined increased resistance of cells to stress-induced oxidative damage as a result of fitness. Forty healthy participants were assigned to either a stress group or a no-stress control group, and measures of stress and oxidative damage were collected. Variation in fitness level across participants was also measured. Oxidative damage increased as a function of stress, but this was not buffered by fitness level. These results should be interpreted with caution because of the small sample size and the limited variability of cardiorespiratory fitness levels in the sample.

Social isolation (SI) is a major risk factor for morbidity and premature mortality in old age. Currently, the physiological and biochemical mechanisms that link SI with adverse health outcomes are unknown. This study examined how SI and age influence mouse memory (novel object recognition (NOR)), anxiety (open field test and elevated plus maze) and hippocampal serotonin (5-HT), 5,hydroxyindole acetic acid (5-HIAA) and norepinephrine (NE), transmitter systems which are important regulators of both behaviours. Male C57BL/6 mice aged 3 or 24 months were housed socially (n = 3) or isolated for 2 weeks prior to experimentation. Following behavioural testing, mice were returned to their home cage for 24 hours and then sacrificed. The hippocampus was dissected for HPLC analysis of NE, 5-HT and 5-HIAA and serum isolated for corticosterone analyses. Age (p

African American women have higher rates of preterm birth (PTB) than women from other racial or ethnic backgrounds. We explored the possibility that African American women experience higher anxiety/lower optimism levels, leading to excess inflammation, a possible pathway leading to PTB. In a cohort of 434 nulliparous women (African American, n = 119; Caucasian, n = 315), standardized measures of anxiety and optimism were completed at 20 weeks’ gestation. C-reactive protein (CRP) was measured in serum collected at the same time, and interleukin-6 (IL-6) was additionally measured in African American women. African American women tended to have higher rates of anxiety ([75th percentile) compared to Caucasian women (27.3 vs. 19.2 %, p = 0.08), but rates of low optimism (

Expression of nitric oxide synthase (NOS) has been found to correlate with tumour progression in breast cancer, indicating that NO activity may drive malignant growth. Previously we have shown that the stress hormone cortisol acts through a nitric oxide synthase (NOS) mediated pathway to induce production of nitric oxide (NO), and can induce DNA damage in breast cancer. Breast cancer cell lines MCF-7 and MDA-MB-231 as well as the mouse mammary tumour cell line 66CL4 were exposed to cortisol and levels of intracellular NO were measured using composite electrochemical sensors. DNA damage was quantified using immunofluorescence and expression of iNOS and metastatic markers VEGF and TWIST were examined using qPCR. An in vivo syngeneic breast cancer model was also used to examine the effect of L-NAME, a NOS inhibitor, on tumour aggressiveness and metastasis in conjunction with daily restraint stress (2hrs) (n=4/group repeated in duplicate). Cortisol significantly increased the expression of iNOS, the generation of NO and DNA damage in breast cancer cells and this was blocked by the NOS inhibitor L-NAME. A significant increase in VEGF and TWIST expression was also observed in response to cortisol. Furthermore, L-NAME also significantly reduced primary tumour growth in stressed mice and reduced the number of metastatic sites/mouse. Tumour microvasculature (as evidenced by CD31 expression) was significantly increased in stressed mice and this was reduced with L-NAME treatment. We demonstrated that L-NAME through inhibition of NO signalling is effective in reducing primary tumour formation and metastatic potential in stressed mice. This data may have impact for patients with breast cancer experiencing extreme stress and further genomic analysis are ongoing.

Esophageal adenocarcinoma (EAC) is associated with a dismal prognosis. The identification of cancer biomarkers can advance the possibility for early detection and better monitoring of tumor progression and/or response to therapy. The authors present results from the development of a serum-based, 4-protein (biglycan, myeloperoxidase, annexin-A6, and protein S100-A9) biomarker panel for EAC.A vertically integrated, proteomics-based biomarker discovery approach was used to identify candidate serum biomarkers for the detection of EAC. Liquid chromatography-tandem mass spectrometry analysis was performed on formalin-fixed, paraffin-embedded tissue samples that were collected from across the Barrett esophagus (BE)-EAC disease spectrum. The mass spectrometry-based spectral count data were used to guide the selection of candidate serum biomarkers. Then, the serum enzyme-linked immunosorbent assay data were validated in an independent cohort and were used to develop a multiparametric risk-assessment model to predict the presence of disease.With a minimum threshold of 10 spectral counts, 351 proteins were identified as differentially abundant along the spectrum of Barrett esophagus, high-grade dysplasia, and EAC (P.05). Eleven proteins from this data set were then tested using enzyme-linked immunosorbent assays in serum samples, of which 5 proteins were significantly elevated in abundance among patients who had EAC compared with normal controls, which mirrored trends across the disease spectrum present in the tissue data. By using serum data, a Bayesian rule-learning predictive model with 4 biomarkers was developed to accurately classify disease class; the cross-validation results for the merged data set yielded accuracy of 87% and an area under the receiver operating characteristic curve of 93%.Serum biomarkers hold significant promise for the early, noninvasive detection of EAC.

BALB/c mice were sensitized and challenged on the skin with the contact sensitizer, dinitroflourobenzene. Acute restraint applied before sensitization diminished, whereas restraint administered before challenge enhanced, chemical-induced ear swelling and leukocytic infiltration in the dermis. Administration of RU486, a glucocorticoid receptor antagonist, partially reversed restraint modulation of the ear swelling response in both restraint paradigms. Restraint did not modulate significantly the concentration of TNF-alpha and IL-1 beta in ear tissue homogenates. These data show that acute restraint modulates cutaneous sensitization differently than challenge, but the changes are not reflected in TNF-alpha or IL-l beta production.

Chemical allergens that induce contact sensitivity cause changes in levels of epidermal cytokines. In mice one of the earliest epidermal cytokines to be upregulated following sensitization is interleukin-1 beta (Iota L-1 beta). The present study investigated the kinetics and in situ localization of induced IL-1 beta expression in mouse skin following topical exposure to the contact allergen oxazolone. Mice were exposed topically to 1% oxazolone, with control mice exposed to vehicle (acetone:olive oil 4:1) alone, and at various times thereafter skin was excised for IL-1 beta mRNA and protein determination by in situ hybridization and enzyme-linked immunosorbant assay (ELISA), respectively. IL-1 beta mRNA was found to be expressed constitutively at low levels in skin from naïve (untreated) and vehicle-treated mice, with mRNA localized in some hair follicles and sebaceous glands; no IL-1 beta mRNA was detected in the epidermis of control animals. Following topical exposure of mice to oxazolone for 5-15 min, upregulation of IL-1 beta mRNA was observed in the epidermis, dermis, hair follicles, and sebaceous glands; at 90 min and beyond the pattern of IL-1 beta mRNA expression declined toward control. Analysis of whole skin homogenates by ELISA demonstrated cutaneous IL-1 beta protein to be present constitutively in both vehicle-treated and naïve mice. Following exposure to oxazolone, cutaneous IL-1 beta protein expression was elevated at 30 min, decreased at 1 h, and fell below the limit of detection of the assay at 2 h before returning to constitutive levels at 4 and 24 h. IL-1 beta protein levels in vehicle-treated mice, naïve mice, and mice treated with the respiratory allergen trimellitic anhydride were unchanged over this time period. The present study demonstrated that IL-1 beta mRNA expression was upregulated rapidly and transiently in well-defined regions of mouse epidermis and dermis during contact sensitization, and was succeeded by an elevation in IL-1 beta protein. This early highly localized upregulation of IL-1 beta lends further support to the hypothesis that this cytokine plays a key role in the initial stages of skin sensitization. Such information will enhance our understanding of the molecular processes involved in allergic contact dermatitis and may provide a mechanistic basis for designing refined animal and in vitro alternatives to existing models of skin sensitization.

Recent studies in rats have indicated that acute restraint enhances cutaneous hypersensitivity. We hypothesized that acute restraint would also modulate the development of allergic and irritant dermatitis in mice and that these restraint-induced changes would be reflected in the cutaneous cytokine profile and be gender-specific. For these studies, male and female B6.129 mice were sensitized and challenged with the contact sensitizer dinitrofluorobenzene or challenged with the irritant croton oil. Two-hour restraint was applied prior to chemical challenge. Restraint combined with chemical increased ear swelling in both genders in ACD, a change that was blocked by administration of RU-486 prior to restraint. Neither restraint nor RU-486 administration modulated development of ICD; however, IL-1beta was decreased by restraint in females only. TNF-alpha and IFN-gamma production were modified in ACD; TNF-alpha in both genders and IFN-gamma in female mice only. Our data demonstrate that acute restraint increases serum corticosterone in B6.129 male and female mice to comparable levels. Restraint modulated the murine ear swelling in ACD, but not ICD, in both genders, and the change in the ear swelling response and cytokine production were, at least in part, corticosterone-dependent.

The induction of contact sensitization and other cutaneous immune responses is dependent upon the activity of epidermal cytokines. One such, keratinocyte-derived tumour necrosis factor alpha (TNF-alpha), is thought to provide the stimulus for the migration of Langerhans cells from the epidermis and their accumulation as immunocompetent dendritic cells in draining lymph nodes. In these investigations we have examined the stimulation by allergen of cutaneous TNF-alpha production and the induced epidermal expression of mRNA for TNF-alpha. Topical exposure of mice to oxazolone, a skin-sensitizing chemical, resulted in cutaneous TNF-alpha protein production that was maximal 2-h following treatment and then declined markedly. The same treatment resulted in highly localized and transient expression of epidermal TNF-alpha mRNA as judged by in situ hybridization. Epidermal mRNA for TNF-alpha was apparent 10 min following exposure to oxazolone, but was no longer detectable at 20 min. A similar pattern of TNF-alpha mRNA expression in the epidermis was provoked by intradermal exposure to interleukin 1 beta, a cytokine shown previously to induce TNF-alpha. Such rigorous regulation of temporal and spatial expression was shown not to be a characteristic of all epidermal cytokines induced by chemical allergen. Exposure to oxazolone under the same conditions resulted in a more widespread and more persistent expression of epidermal mRNA for interleukin 6. These data demonstrate that during skin sensitization the induced expression of epidermal TNF-alpha is finely controlled in space and time. It is proposed that such regulation facilitates the initiation of cutaneous immune responses while preventing excessive inflammation that would result from more persistent TNF-alpha production.

Stress triggers complex response mechanisms designed to recognize and adapt to perturbations in homeostasis. The immune system is highly responsive to stress, although the complete mechanisms linking stress and immune mediators including T lymphocytes, are not fully understood. Stress exerts its effects on immune effectors through two primary pathways: the sympathetic-adrenal-medullary pathway, and the hypothalamic-pituitary-adrenal pathway which modulate adaptive immunity and lymphocyte migration. In this report we show that stress via release of stress hormones induces early T cell activation and greatly impacts the cytoskeleton by modulating numerous actin-regulating proteins. In particular, proteomic profiling revealed significant decreases in numerous key actin-binding proteins including moesin. Although confocal microscopy showed that moesin and actin were uniformly distributed on the surface of resting T cells, a remarkable polarization and redistribution of moesin and actin was observed following treatment with stress hormones with moesin localizing at the distal pole complex. In addition, the alteration in moesin localization and eventual decrease in expression were accompanied by a loss of CD43; a receptor involved in negatively regulating T cell activation. In conclusion, we have defined a novel molecular mechanism whereby stress hormones negatively impact T cell activation and migration through regulation of key cytoskeletal and plasma membrane factors.

Breast cancer is a highly heterogeneous disease, an observation that underscores the importance of elucidating conserved molecular characteristics, such as gene and protein expression, across breast cancer cell types toward providing a greater understanding of context-specific features central to this disease. Motivated by the goal of defining central biological themes across breast cancer cell subtypes, we conducted a global proteomic analysis of three breast cancer cell lines, MCF7, SK-BR-3, and MDA-MB-231, and compared these to a model of nontransformed mammary cells (MCF10A). Our results demonstrate modulation of proteins localized to the extracellular matrix, plasma membrane, and nucleus, along with coordinate decreases in proteins that regulate "cell spreading," a cellular event previously shown to be dysregulated in transformed cells. Protein interaction network analysis revealed the clustering of focal adhesion kinase (FAK), a fundamental regulator of cell spreading, with several proteins identified as mutually, differentially abundant across breast cancer cell lines that impact expression and activity of FAK, such as neprilysin and keratin 19. These analyses provide insights into conservation of protein expression across breast cancer cell subtypes, a subset of which warrants further investigation for their roles in the regulation of cell spreading and FAK in breast cancer.

Tissue interstitial fluid (TIF) bathes cells in tissues, and it is hypothesized that TIF proximal to a developing tumor may contain an enriched population of tumor-specific shed and secreted proteins relative to peripheral blood. Extraction of TIF proteins is typically accomplished through passive incubation of surgically resected tissues in phosphate buffered saline (PBS); however, its influence on cellular activity and viability has not been fully explored. The present investigation sought to characterize whether different buffer systems influence the recovered TIF proteome. Five TIF buffer systems were investigated including PBS, Dulbecco's modified Eagle medium (DMEM), and three organ transplantation preservative solutions: Celsior solution S (CS), histidine-tryptophan-ketoglutarate (HTK), and University of Wisconsin (UW). Kidney tumor, adjacent normal kidney, and ovarian tumor tissues were incubated in each of the buffer systems, and the harvested TIF proteins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the present results indicate that no significant differences exist in the recovered proteins from these two neoplasms between the five solution groups, additional sample preparative steps are required prior to LC-MS/MS for TIF proteins harvested from DMEM, UW, CS, and HTK. These data support that PBS is a suitable and convenient solution for harvesting TIF proteins for MS-based proteomics.

Chemotherapy comprises part of successful treatment regimens for breast cancer, however, up to 50% of patients develop resistance. Stress in cancer patients can equate to poor chemotherapeutic responses. We hypothesize that drug resistance may be associated with stress hormone-induced alterations in breast cancer cells. To test this hypothesis, MDA-MB-231 cells were cultured with paclitaxel and/or cortisol, norepinephrine and epinephrine and cytotoxicity, cell cycle analyses, genomic and proteomic analyses were performed. Paclitaxel-mediated cytotoxicity and G2/M cell cycle arrest were reversed significantly by stress hormones. Genomic and proteomic analyses revealed that stress hormones modulated beta-tubulin isotypes and significantly altered genes and proteins involved in regulation of the G2/M transition, including cyclin-dependent kinase-1 (CDK-1). Inhibition of CDK-1 abrogated stress hormone-mediated reversal of paclitaxel-induced cytotoxicity, indicating that the protective effect of stress hormones act through a CDK-1-dependent mechanism. These data demonstrate that stress hormones interfere with paclitaxel efficacy and contribute significantly to drug resistance.

Stress is associated with increased production of sympathetic and other adrenal hormones. Epinephrine (E), norepinephrine (NE) and cortisol are produced during psychological stress and may affect many cells directly. These effects may be transient (e.g. heart rate, immune cell trafficking) or they can have more long-lasting consequences, such as permanent DNA damage which may result in increased cell transformation and/or tumorigenicity. Here, the molecular effects of short term in vitro exposure of these stress hormones were analyzed on murine 3T3 cells by measuring effects on DNA damage and repair, cell transformation and changes in mRNA expression of genes specifically involved in DNA damage signaling pathways. Short-term exposure (

Chronic beryllium disease (CBD) is a debilitating, granulomatous lung disease that occurs in 1 to 5% of exposed workers. Beryllium stimulates a major histocompatibility Class II-restricted, TH1, CD4+ T cell-mediated immune response. The immunological component of the illness, coupled with the small subset of beryllium workers who develop disease, led researchers to hypothesize that genetic factors modify risk of disease. Analysis of human leukocyte antigen (HLA) genes, the T cell receptor, and tumor necrosis factor (TNF)-alpha focused on three critical steps in the development of beryllium specific immunity. Molecular epidemiological analysis of the association of HLA-DP, -DR, and -DQ has implicated HLA-DPB1E69 allelic variants in disease; however, its role in sensitization is unclear. A single report suggested association between HLA-DQB1G86 and progression from sensitization to disease. A beryllium-specific binding motif was identified in CBD-derived T cell clones. Beryllium-stimulated proliferation using HLA-DPB1*0201 and TCRAV22S1/TCRBVb3 T cell receptors (TCRs) confirmed beryllium specificity of these molecules. The G/A transition at -308 in the TNF-alpha promoter was associated with high concentrations of TNF-alpha in bronchoalveolar lavage and to disease severity. Although these studies are continuing, the data confirm the role of genetic factors in the cellular response to beryllium.

DNA damage-, DNA repair-, and apoptosis-related gene expression in CD3 + T lymphocytes of BALB/c mice subjected to 2-h restraint stress were compared to that in CD3 + T lymphocytes from control mice. Using targeted cDNA arrays, significant increases in expression of genes serving as sensors of DNA damage, including MSH genes and RAD53, were observed. GADD45g, a gene responsible for regulating cell cycle arrest and apoptosis, was significantly induced; as was Pura, a gene involved in cell proliferation. These data suggest that, at the molecular level, stress activates genes responsible for priming the T cell to either undergo apoptosis or proliferation. D 2005 Elsevier B.V. All rights reserved.

Exposure of skin to noxious environmental stimuli can cause allergic contact dermatitis (ACD), which is a major health risk. Epidemiological studies have determined that 40% of workers report that their jobs are very, or extremely, stressful, and the number of chemicals to which workers are exposed increases each year. We hypothesized that combined exposure to a workplace stressor and a sensitizing chemical would alter the time course and magnitude of the skin immune response. We assessed the mixed exposure of chemical and restraint stress using three potent skin sensitizers, 2,4 dinitrofluorbenzene (DNFB), dicyclohexylcarbodiimide (DCC), and oxazolone, (OXA) on the ear swelling response in stress-susceptible BALB/c mice. Quantitative analyses showed that the dose–response relationship for each chemical followed a cubic trend. Although stress did not alter the shape of the curve, application of restraint stress on day 1 or on day 6 diminished the ear swelling response to 0.1% DNFB. However, if the concentration of the challenge dose was increased to a more irritating concentration, 0.25% DNFB, ear swelling was enhanced. Restraint stress applied on day 6 also increased ear swelling in response to the highly irritating sensitizer DCC, but not to the low-irritancy chemical OXA. These data support the hypothesis that dose–response relationships exist for sensitization with chemical and that restraint stress modulation of the ear swelling response is both chemical specific and dependent on the irritancy potential of the chemical.

C57BL/6 mice, in contrast to BALB/c mice, display minimal behavioral changes in response to environmental stressors and are considered relatively stress-resistant. We have shown that application of acute restraint prior to chemical challenge enhanced cutaneous hypersensitivity (CHS) in BALB/c mice and that this enhanced response is partially glucocorticoid dependent. Due to strain differences in the immune response and in the response to environmental stressors, we hypothesized that acute restraint would not enhance CHS in the less stress-sensitive C57BL/6 mice. We sensitized and challenged C57BL/6 mice with the contact sensitizer, 2, 4-dinitrofluorobenzene (DNFB) in the presence and absence of restraint. Acute restraint, applied prior to chemical challenge, significantly increased serum corticosterone, but to concentrations approximately 60% of those reported for BALB/c mice. Neither restraint nor the exogenous administration of corticosterone enhanced chemical-induced ear swelling in C57BL/6 mice. Pharmacological interruption of the hypothalamic pituitary adrenal axis (HPAA) with the glucocorticoid type II receptor antagonist, RU486, did not alter the development of CHS, however, adrenalectomized (ADX) mice exhibited decreased ear swelling, a measurement that was decreased further by restraint. Combined application of acute restraint and corticosterone prior to chemical challenge significantly enhanced the ear swelling response in C57BL/6 wild-type mice. These data confirm that C57BL/6 mice have a blunted corticosterone response to restraint and that acute restraint does not modulate cutaneous hypersensitivity. Furthermore, our data demonstrate that stress-resistance is not conferred exclusively through the glucocorticoid pathways.