Is breast best? Perceptions of infant feeding

This study explored the reasons why young women from low income areas are among those least likely to breastfeed. Focus groups were conducted with 15 health professionals and 11 young, first time mothers were interviewed. Health professionals participating believed that white communities endorsed bottle feeding while Pakistani and Bangladeshi communities, although they accepted breastfeeding more readily, were likely to give prelacteal feeds of non-breast milk and to delay weaning. The interviews with mothers revealed a belief that 'breast is best' but factors intervened in a detrimental way resulting in the decision not to breastfeed or in early cessation. Participating mothers expected breastfeeding to be painful and were preoccupied with feeding and weight gain. The desire to have 'fat bonnie babies' demonstrated the mothers' moral attempts to be perceived as 'good mothers' although their actions went against the knowledge that 'breast is best'. Recommendations include educating health professionals about subcultures in their communities and reversing the misconception that breast milk is insufficient for a baby's healthy development. Promoting breastfeeding must include the crucial message that breast milk contains all the nourishment a baby needs.

Neonatal enteral feeding tubes as loci for colonisation by members of the Enterobacteriaceae

Background The objective of this study was to determine whether neonatal nasogastric enteral feeding tubes are colonised by the opportunistic pathogen Cronobacter spp. (Enterobacter sakazakii) and other Enterobacteriaceae, and whether their presence was influenced by the feeding regime. Methods One hundred and twenty-nine tubes were collected from two neonatal intensive care units (NICU). A questionnaire on feeding regime was completed with each sample. Enterobacteriaceae present in the tubes were identified using conventional and molecular methods, and their antibiograms determined. Results The neonates were fed breast milk (16%), fortified breast milk (28%), ready to feed formula (20%), reconstituted powdered infant formula (PIF, 6%), or a mixture of these (21%). Eight percent of tubes were received from neonates who were 'nil by mouth'. Organisms were isolated from 76% of enteral feeding tubes as a biofilm (up to 107 cfu/tube from neonates fed fortified breast milk and reconstituted PIF) and in the residual lumen liquid (up to 107 Enterobacteriaceae cfu/ml, average volume 250 µl). The most common isolates were Enterobacter cancerogenus (41%), Serratia marcescens (36%), E. hormaechei (33%), Escherichia coli (29%), Klebsiella pneumoniae (25%), Raoultella terrigena (10%), and S. liquefaciens (12%). Other organisms isolated included C. sakazakii (2%),Yersinia enterocolitica (1%),Citrobacter freundii (1%), E. vulneris (1%), Pseudomonas fluorescens (1%), and P. luteola (1%). The enteral feeding tubes were in place between < 6 h (22%) to > 48 h (13%). All the S. marcescens isolates from the enteral feeding tubes were resistant to amoxicillin and co-amoxiclav. Of additional importance was that a quarter of E. hormaechei isolates were resistant to the 3rd generation cephalosporins ceftazidime and cefotaxime. During the period of the study, K. pneumoniae and S. marcescens caused infections in the two NICUs. Conclusion This study shows that neonatal enteral feeding tubes, irrespective of feeding regime, act as loci for the bacterial attachment and multiplication of numerous opportunistic pathogens within the Enterobacteriaceae family. Subsequently, these organisms will enter the stomach as a bolus with each feed. Therefore, enteral feeding tubes are an important risk factor to consider with respect to neonatal infections.

An aqueous extract of Fagonia cretica induces DNA damage, cell cycle arrest and apoptosis in breast cancer cells via FOXO3a and p53 expression

Background - Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings - Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as ?-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance - Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression.

Heparanase and COX-2 expression as predictors of lymph node metastasis in large, high-grade breast tumors

Background/Aim: Heparanase (HPA) contributes to breast cancer metastasis by facilitating the breakdown of the basement membrane and extracellular matrix. High expression of HPA is thought to be associated with increased nodal involvement and poor survival in patients with breast cancer. Overexpression of cyclooxygenase-2 (COX-2) in breast cancer is associated with indicators of poor prognosis such as lymph node metastasis, poor differentiation, and large tumor size. The underlying mechanism by which HPA and COX-2 overexpression increases the metastatic potential of breast cancer is not fully-understood. To enhance our understanding over these mechanisms, we aimed to investigate the relationship between the size of the tumor and HPA expression, tumor grade as well as lymph node status in patients with breast cancer. Materials and Methods: Immunohistochemical analysis of HPA and COX-2 expression was performed on 246 breast tumor samples. The expression of HPA was correlated with COX-2 expression, tumor grade, lymph node status, oestrogen receptor status. Results: The overexpression of HPA and COX-2 was associated with increased likelihood of lymph node positivity in large, high-grade tumors. High-grade tumors with size greater than 20 mm, that overexpressed HPA, were 4-times more likely to be associated with lymph node involvement (OR 4.71, CI 1.21-18.25). Whereas, tumors greater than 20 mm in size were 5-times more likely to metastasize to the regional lymph nodes, if associated with overexpression of COX-2 (OR 5.5, CI 1.2-24.8). Conclusion: Expression of HPA appears to be a key mechanism by which large, highgrade breast tumors metastasize to regional lymph nodes, while COX-2 overexpression may be an independent predictor of lymph node positivity.

Novel expression of microRNAs in serum samples of Iraqi breast cancer women

Although a lot of hard work against cancer to reduces its spread but it still continues to kill with abandon. The need for a biomarker for cancer early detection becomes the most mind concentrated scientists. MicroRNAs the tiny non coding RNA molecules opened new path for the scientists to determine the cancer in its early stages. Expression of microRNAs profiles has been investigated to be involved in cancer development. Here we determined the expression of microRNAs in serum of Iraqi healthy volunteers and other women diagnosed with breast cancer. MicroRNAs expression has been determined by using real time qPCR and delta method has been used. Four of thirteen microRNAs were shown to be expressed in serum of Iraqi breast cancer women. Let-7a and miR-21 were shown to be significantly over expressed in serum of breast cancer compared with healthy serum volunteers (P= 0.022 and 0.026) respectively. While miR-26b and miR-429 found to be significantly down expressed in serum of breast cancer women (P= 0.0034 and 0.031) respectively. The result concluded that these expressed microRNAs in serum of breast cancer women could be used as a first indicator of breast cancer occurrence.

Phytoestrogens modulate breast cancer resistance protein expression and function at the blood-cerebrospinal fluid barrier

PURPOSE: Breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter expressed at the blood cerebrospinal fluid barrier (BCSFB), and influences distribution of drugs into the central nervous systems (CNS). Current inhibitors have failed clinically due to neurotoxicity. Novel approaches are needed to identify new modulators to enhance CNS delivery. This study examines 18 compounds (mainly phytoestrogens) as modulators of the expression/function of BCRP in an in vitro rat choroid plexus BCSFB model. METHODS: Modulators were initially subject to cytotoxicity (MTT) assessment to determine optimal non-toxic concentrations. Reverse-transcriptase PCR and confocal microscopy were used to identify the presence of BCRP in Z310 cells. Thereafter modulation of the intracellular accumulation of the fluorescent BCRP probe substrate Hoechst 33342 (H33342), changes in protein expression of BCRP (western blotting) and the functional activity of BCRP (membrane insert model) were assessed under modulator exposure. RESULTS: A 24 hour cytotoxicity assay (0.001 µM-1000 µM) demonstrated the majority of modulators possessed a cellular viability IC50 > 148 µM. Intracellular accumulation of H33342 was significantly increased in the presence of the known BCRP inhibitor Ko143 and, following a 24 hour pre-incubation, all modulators demonstrated statistically significant increases in H33342 accumulation (P < 0.001), when compared to control and Ko143. After a 24 hour pre-incubation with modulators alone, a 0.16-2.5-fold change in BCRP expression was observed for test compounds. The functional consequences of this were confirmed in a permeable insert model of the BCSFB which demonstrated that 17-β-estradiol, naringin and silymarin (down-regulators) and baicalin (up-regulator) can modulate BCRP-mediated transport function at the BCSFB. CONCLUSION: We have successfully confirmed the gene and protein expression of BCRP in Z310 cells and demonstrated the potential for phytoestrogen modulators to influence the functionality of BCRP at the BCSFB and thereby potentially allowing manipulation of CNS drug disposition.

Phytochemical mediated-modulation of the expression and transporter function of breast cancer resistance protein at the blood-brain barrier:an in-vitro study

Clinical translation of BCRP inhibitors have failed due to neurotoxicity and novel approaches are required to identify suitable modulators of BCRP to enhance CNS drug delivery. In this study we examine 18 compounds, primarily phytochemicals, as potential novel modulators of AhR-mediated regulation of BCRP expression and function in immortalised and primary porcine brain microvascular endothelial cells as a mechanism to enhance CNS drug delivery. The majority of modulators possessed a cellular viability IC50 > 100 µM in both cell systems. BCRP activity, when exposed to modulators for 1 hour, was diminished for most modulators through significant increases in H33342 accumulation at < 10 µM with 2,6,4-trimethoflavone increasing H33342 intracellular accumulation by 3.7–6.6 fold over 1–100 µM. Western blotting and qPCR identified two inducers of BCRP (quercetin and naringin) and two down-regulators (17-β-estradiol and curcumin) with associated changes in BCRP efflux transport function further confirmed in both cell lines. siRNA downregulation of AhR resulted in a 1.75 ± 0.08 fold change in BCRP expression, confirming the role of AhR in the regulation of BCRP. These findings establish the regulatory role AhR of in controlling BCRP expression at the BBB and confirm quercetin, naringin, 17-β-estradiol, and curcumin as novel inducers and down-regulators of BCRP gene, protein expression and functional transporter activity and hence potential novel target sites and candidates for enhancing CNS drug delivery.

Expression of the human cachexia-associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia-associated protein (HCAP) was recently identified from a breast cancer library based on the available 20-amino acid sequence of proteolysis-inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP-associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ-confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC-3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia. © 2003 Wiley-Liss, Inc.

Predictive gene lists for breast cancer prognosis: a topographic visualisation study

Background: The controversy surrounding the non-uniqueness of predictive gene lists (PGL) of small selected subsets of genes from very large potential candidates as available in DNA microarray experiments is now widely acknowledged 1. Many of these studies have focused on constructing discriminative semi-parametric models and as such are also subject to the issue of random correlations of sparse model selection in high dimensional spaces. In this work we outline a different approach based around an unsupervised patient-specific nonlinear topographic projection in predictive gene lists. Methods: We construct nonlinear topographic projection maps based on inter-patient gene-list relative dissimilarities. The Neuroscale, the Stochastic Neighbor Embedding(SNE) and the Locally Linear Embedding(LLE) techniques have been used to construct two-dimensional projective visualisation plots of 70 dimensional PGLs per patient, classifiers are also constructed to identify the prognosis indicator of each patient using the resulting projections from those visualisation techniques and investigate whether a-posteriori two prognosis groups are separable on the evidence of the gene lists. A literature-proposed predictive gene list for breast cancer is benchmarked against a separate gene list using the above methods. Generalisation ability is investigated by using the mapping capability of Neuroscale to visualise the follow-up study, but based on the projections derived from the original dataset. Results: The results indicate that small subsets of patient-specific PGLs have insufficient prognostic dissimilarity to permit a distinction between two prognosis patients. Uncertainty and diversity across multiple gene expressions prevents unambiguous or even confident patient grouping. Comparative projections across different PGLs provide similar results. Conclusion: The random correlation effect to an arbitrary outcome induced by small subset selection from very high dimensional interrelated gene expression profiles leads to an outcome with associated uncertainty. This continuum and uncertainty precludes any attempts at constructing discriminative classifiers. However a patient's gene expression profile could possibly be used in treatment planning, based on knowledge of other patients' responses. We conclude that many of the patients involved in such medical studies are intrinsically unclassifiable on the basis of provided PGL evidence. This additional category of 'unclassifiable' should be accommodated within medical decision support systems if serious errors and unnecessary adjuvant therapy are to be avoided.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The Plasma Membrane Calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, up-regulation in the expression of the pro-apoptotic protein Fas Ligand, and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

Cytotoxic activity of Fagonia cretica against human breast cancer cells

In many parts of the world, plants are directly utilised for their medicinal properties. Traditional medicine from Pakistan, India and the Far East is well documented and its history is embedded in folklore. It has been documented that an aqueous extract of the desert shrub, Fagonia cretica, is a popular treatment for breast cancer in Pakistan. The administration of an aqueous extract of Fagonia cretica is reported effective at reducing tumour size and improving the quality of life of breast cancer patients, is well tolerated and does not exhibit adverse effects like vomiting, diarrhoea or alopecia which are common side effects of standard cytotoxic therapy. In the past, many pharmacologically active and chemotherapeutic compounds have been isolated from plants which subsequently have proven to be successful in clinical trials and been used as primary compounds in therapeutic regimes. Fagonia cretica has historical use as a treatment for breast cancer, yet there is little scientific evidence which shows chemotherapeutic potential towards breast tumours. Preparation and analysis of an aqueous extract of Fagonia cretica may reveal novel chemotherapeutic agents that can be used to effectively target cancer cells. An understanding of the mechanism of any activity may improve our understanding of cancer cell biology and reveal novel therapeutic targets. This thesis describes for the first time that an aqueous extract of Fagonia cretica shows potent in vitro cytotoxic activity towards breast cancer epithelial cell lines which was not seen towards normal mammary epithelial cells. Elucidation and characterisation of the cytotoxic mechanism was undertaken by analysing DNA damage, cell cycle status, apoptosis, metabolic state and expression of transcription factors and their targets. Finally, methods for the isolation and identification of active compound(s) were developed using various chromatographic techniques. An aqueous extract of Fagonia cretica was able to reduce cell viability significantly in two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231). This activity was markedly reduced in normal mammary epithelial cells (HMEpC). Further investigation into the mode of action revealed that extract treatment induced cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cell lines. This coincided with the formation of DNA double stranded breaks and the DNA repair marker ?-H2AX. In MCF-7 cells, ATM/ATR activation resulted in increased p53 expression and of its transcriptional targets p21 and bax, suggesting a role for a p53-mediated response. Furthermore, inhibition of extract-induced p53 expression with siRNA reduced the cytotoxic effect against MCF-7 cells. Extract treatment was also associated with increased FOXO3a expression in MCF-7 and MDA-MB-231 cells. In the absence of functional p53, siRNA knockdown of extract-induced FOXO3a expression was completely abrogated, suggesting that FOXO3a plays a vital role in extract-induced cytotoxicity. Isolation and characterisation of the active compound(s) within the extract was attempted using liquid chromatography and mass spectrometry in conjunction with a cell viability assay. Multiple fractionations generated an active fraction that contained four major compounds as detected by mass spectrometry. However, none of these compounds were identified structurally or chemically due to constraints within the methodology.

Tissue Transglutaminase (TG2) is a potential therapeutic target in the treatment of chemoresistant breast cancer

Tissue transglutaminase (TG2) has been suggested to be a key player in the progression and metastasis of chemoresistant breast cancer. One of the foremost survival signalling pathways implicated in causing drug resistance in breast cancer is the constitutive activation of NFκB (Nuclear Factor -kappa B) induced by TG2. This study provides a mechanism by which TG2 constitutively activates NFκB which in turn confers chemoresistance to breast cancer cells against doxorubicin. Breast cancer cell lines with varying expression levels of TG2 as well as TG2 null breast cancer cells transfected with TG2 were used as the major cell models for this study. This study made use of cell permeable and impermeable TG2 inhibitors, specific TG2 and Rel A/ p65 targeting siRNA, TG2 functional blocking antibodies, IKK inhibitors and a specific targeting peptide against Rel A/p65 to investigate the pathway of activation involved in the constitutive activation of NFκB by TG2 leading to drug resistance. Crucial to the activation of Rel A/p65 and drug resistance in the breast cancer cells is the interaction between the complex of IκBα and Rel A/p65 with TG2 which results in the dimerization of Rel A/p65 and polymerization of IκBα. The association of TG2 with the IκBα-NFκB complex was determined to be independent of IKKα/β function. The polymerized IκBα is degraded in the cytoplasm by the μ-calpain pathway which allows the cross linked Rel A/ p65 dimers to translocate into the nucleus. Using R283 and ZDON (cell permeable TG2 activity inhibitors) and specific TG2 targeting siRNA, the Rel A/ p65 dimer formation could be inhibited. Co-immunoprecipitation studies confirmed that the phosphorylation of the Rel A/p65 dimers at the Ser536 residue by IKKε took place in the cell nucleus. Importantly, this study also investigated the transcriptional regulation of the TGM2 gene by the pSer536 Rel A/ p65 dimer and the importance of this TG2-NFκB feedback loop in conferring drug resistance to breast cancer cells. This data provides evidence that TG2 could be a key therapeutic target in the treatment of chemoresistant breast cancer.

The Role of Aquaporin 3 (AQP3) in Breast Cancer

The increasing prevalence of breast cancer (BC) in different parts of the world, particularly in the UK, highlights the importance of research into the aetiology and pathology of the disease. BC is the most common malignancy affecting women worldwide. Aquaporins (AQPs) are membrane protein channels that regulate cellular water flow. Recently, studies have demonstrated that expression of AQP3 is up-regulated in cancerous breast tissue. The present study examines the role of AQP3 in BC cell biology. Examination of clinical cases of BC showed higher AQP3 gene and protein expression in cancer tissues compared to healthy border tissues. In distinct clinicopathological groups however there were no differences observed with regards to AQP3 expression, suggesting that AQP3 expression may not be a predictor of lymph node infiltration or tumour grade. shRNA technology was used to knockdown gene expression of AQP3 in the invasive MDA-MB-231 BC cellular model. Cellular proliferation, migration, invasion, adhesion and response to the 5- fluorouracil (5-FU) based chemotherapy treatment were investigated in parental and knockdown cell line. AQP3 knockdown cells showed reduction in cellular proliferation, migration, invasion and increase in cell sensitivity to 5-FU compared with wild type (WT) or scrambled control (SC) cells. The effects of AQP3 knockdown on cellular glycolytic ability and ATP cellular content were quantified. Indirect glucose uptake was also measured by quantifying reconditioned media. AQP3 knockdown cells showed significantly lower levels of glucose uptake as compared to WT or SC. However there was no difference in the glycolytic ability and ATP content of the cells suggesting AQP3 has no role in cancer cell energetics. These data collectively suggest AQP3 expression is associated with the BC disease clinically and plays a role in multiple important aspects of BC pathophysiology, thus AQP3 represents a novel target for therapeutic intervention.

Evaluating the evidence for targeting FOXO3a in breast cancer:a systematic review

Background: Tumour cells show greater dependency on glycolysis so providing a sufficient and rapid energy supply for fast growth. In many breast cancers, estrogen, progesterone and epidermal growth factor receptor-positive cells proliferate in response to growth factors and growth factor antagonists are a mainstay of treatment. However, triple negative breast cancer (TNBC) cells lack receptor expression, are frequently more aggressive and are resistant to growth factor inhibition. Downstream of growth factor receptors, signal transduction proceeds via phosphatidylinositol 3-kinase (PI3k), Akt and FOXO3a inhibition, the latter being partly responsible for coordinated increases in glycolysis and apoptosis resistance. FOXO3a may be an attractive therapeutic target for TNBC. Therefore we have undertaken a systematic review of FOXO3a as a target for breast cancer therapeutics. Methods: Articles from NCBI were retrieved systematically when reporting primary data about FOXO3a expression in breast cancer cells after cytotoxic drug treatment. Results: Increased FOXO3a expression is common following cytotoxic drug treatment and is associated with apoptosis and cell cycle arrest. There is some evidence that metabolic enzyme expression is also altered and that this effect is also elicited in TNBC cells. FOXO3a expression serves as a positive prognostic marker, especially in estrogen (ER) receptor positive cells. Discussion: FOXO3a is upregulated by a number of receptor-dependent and -independent anti-cancer drugs and associates with apoptosis. The identification of microRNA that regulate FOXO3a directly suggest that it offers a tangible therapeutic target that merits wider evaluation.