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ABSTRACT: Aluminium is not a physiological component of the breast but has been measured recently in human breast tissues and breast cyst fluids at levels above those found in blood serum or milk. Since the presence of aluminium can lead to iron dyshomeostasis, levels of aluminium and iron-binding proteins (ferritin, transferrin) were measured in nipple aspirate fluid (NAF), a fluid present in the breast duct tree and mirroring the breast microenvironment. NAFs were collected noninvasively from healthy women (NoCancer; n = 16) and breast cancer-affected women (Cancer; n = 19), and compared with levels in serum (n = 15) and milk (n = 45) from healthy subjects. The mean level of aluminium, measured by ICP-mass spectrometry, was significantly higher in Cancer NAF (268.4 ± 28.1 μg l(-1) ; n = 19) than in NoCancer NAF (131.3 ± 9.6 μg l(-1) ; n = 16; P < 0.0001). The mean level of ferritin, measured through immunoassay, was also found to be higher in Cancer NAF (280.0 ± 32.3 μg l(-1) ) than in NoCancer NAF (55.5 ± 7.2 μg l(-1) ), and furthermore, a positive correlation was found between levels of aluminium and ferritin in the Cancer NAF (correlation coefficient R = 0.94, P < 0.001). These results may suggest a role for raised levels of aluminium and modulation of proteins that regulate iron homeostasis as biomarkers for identification of women at higher risk of developing breast cancer. The reasons for the high levels of aluminium in NAF remain unknown but possibilities include either exposure to aluminium-based antiperspirant salts in the adjacent underarm area and/or preferential accumulation of aluminium by breast tissues.
Journal of Applied Toxicology 02/2011; 31(3):262-9. · 2.48 Impact Factor
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ABSTRACT: Oxygen is a fundamental element for all living organisms, and modifications in its concentration influence several physiological and pathological events such as embryogenesis, development and also aging. Regulation of oxygen levels is an important factor in neural stem cell biology (e.g. differentiation, growth and the capacity to generate more differentiated cells). Studies on neural stem cells in culture have deepened our knowledge of their survival, proliferation and differentiation pathways. However, traditional cell culture for neural stem cells is performed employing environmental oxygen levels of 20%, while the effective oxygen concentration in the developing and adult brain is significantly lower; this results in an important alteration of the in vivo conditions. Several data indicate that a so called "physiologic hypoxic condition" could strongly influence the growth of neural stem cells and their differentiation mechanisms both in vivo and in vitro. The present overview deals with the different mechanisms utilized by invertebrate and vertebrate organisms to respond to hypoxic conditions. It highlights how the adaptations and responses to different oxygen concentrations have changed along the developmental route and underlines the importance of oxygen concentration in neural physiology and differentiation, with a final hint to the involvement of hypoxia in brain cancer stem cells.
The International journal of developmental biology 01/2011; 55(6):569-81. · 2.16 Impact Factor
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ABSTRACT: Analysis of nipple aspirate fluid (NAF) allows the noninvasive identification of both cellular and biomolecular markers of human breast cancer, the most common female malignancy in developed countries. Cytosolic superoxide dismutase (SOD-1) represents a detoxifying enzyme able to regulate the balance between oncogenic and oncosuppressor reactive oxygen species.
We analyzed SOD-1 expression in 126 NAF samples collected from 67 women with and 59 without breast cancer, using enzyme-linked immunosorbent assay, immunoprecipitation, Western blotting, and immunocytochemistry.
SOD-1 median values in plasma presented no difference between the no-cancer and cancer subgroups. No significant difference in the median level of SOD-1 between matched plasma and NAF from cancer patients was found, whereas SOD-1 median level in no-cancer NAF was significantly higher when compared with matched plasma. Finally, the SOD-1 median value in no-cancer NAF was significantly higher (about 2-fold) than in cancer NAF.
NAF measurement of SOD-1 is a useful tool to identify intracellular redox status of the breast microenvironment, mirroring the oxidative metabolic pathways occurring in breast tissue, both in physiologic and cancer conditions and in improving the identification of women at increased breast cancer risk. SOD-1 activity in the breast microenvironment may represent a functional "switch" between the detrimental/oncogenic properties and the oncosuppressor/proapoptotic role of this antioxidant enzyme.
Clinical Breast Cancer 06/2010; 10(3):238-45. · 2.38 Impact Factor
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ABSTRACT: Breast cancer, a worldwide disease with increasing incidence, develops from ductal/lobular epithelium. Nipple aspirate fluid (NAF), secreted from the breast ducts and lobules, can be analyzed to assess metabolic activity in breast microenvironment. Premalignant and malignant cell alterations may produce biochemical signals that deliver inflammatory proteins to the site. C-reactive protein (CRP), acute-phase protein considered a prognostic marker of inflammation, is frequently over-expressed in invasive breast carcinomas. Starting from the evidence that soluble and cell-bound iron binding protein Ferritin (FTN) and Transferrin (TRF) are crucially involved in breast inflammation and cancer, the aim of the present study is to analyze in NAF (a ductal fluid mirroring the breast microenvironment noninvasively collected from healthy and proven breast cancer affected women, n=38), the concentrations of CRP, FTN and TRF through high sensitive immunoassays. We analysed also serum (n=35) and milk samples (n=20) from healthy subjects. The mean level of CRP in Cancer NAF was significantly higher than in NoCancer NAF (P < 0.0001), especially in postmenopausal patients. Moreover, in Cancer NAF we detected higher levels of TRF and FTN respect to NoCancer NAF (P<0.001). A highly significant positive correlation between FTN and CRP content (Y= 2322x + 6.196, r(2) = 0.651, P<0.0001) was found. These data may support the involvement of inflammation and deregulation of iron homeostasis in breast cancer etio-pathogenesis. The significant accumulation of CRP in NAF in conjunction to the disruption of iron homeostasis may help to identify women at higher breast cancer risk.
American Journal of Translational Research 01/2010; 3(1):100-13.
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ABSTRACT: Breast cancer, a complex and multifactorial disease, is the most commonly diagnosed malignancy affecting women. Methods currently available for breast cancer detection have well-described limitations; in this respect, the intraductal approaches directly assess the microenvironment of the breast. Nipple aspirate fluid (NAF) can be noninvasively obtained from the breast in most women and represents a promising biological tool to assess metabolic, hormonal and molecular changes occurring in the cells lining the ducts, from which breast cancer arises. The aim of this review is to highlight the application of NAF studies in the field of biomarker discovery, which provide results useful for early detection and prevention of breast cancer risk; in fact, the analysis of NAF (mirroring the ductal-lobular microenvironment) is a reliable method for assessment of metabolic/hormonal pathways within the mammary gland, identifying biomolecular mechanisms of breast cancer initiation and progression. The intracrinology of breast microenvironment (i.e., hormonal status in NAF) may provide independent diagnostic/prognostic factors, highlighting the importance of early altered hormonal metabolism (e.g., aromatase, estrogen sulfotransferase and steroid sulfatase pathway) in relation to breast cancer initiation. The possible application of targeted therapies through the inhibition of intratumoral enzymes involved in steroid metabolism is also discussed. The intraductal approach to hormone analyses may provide a further panel of biomarkers providing clinical benefits and strengthening the armory against breast cancer.
Expert Review of Endocrinology & Metabolism 08/2009; 4(5):493-505.
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ABSTRACT: Protein and proteomic high-throughput technologies provide the polypeptide signatures of nipple aspirate fluid (NAF), a breast secretion collected noninvasively from healthy individuals and cancer patients. As breast cancer develops from ductal-lobular epithelium, the analysis of NAF (mirroring the ductal-lobular microenvironment) is a useful tool for the analysis of metabolic pathways within the mammary gland, deepening our knowledge of the biomolecular mechanisms of breast cancer initiation and progression. The different protein expression of major NAF proteins, separated using 1D polyacrylamide gels, has proven valuable for the early detection of women with increased risk of cancer. The failure to recognize a single marker with sufficient clinical sensitivity and/or specificity has driven the identification of breast cancer multiple proteins by 2D electrophoresis. Mass spectrometry-based proteomic approaches (SELDI- and MALDI-TOF technologies) have allowed the characterization of differential NAF proteomic fingerprints between healthy individuals and breast cancer patients. The intraductal approach of protein and proteomic analyses may provide a panel of biomarkers to strengthen the armory against breast cancer.
Expert Review of Proteomics 03/2009; 6(1):43-60. · 3.68 Impact Factor
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ABSTRACT: Matrix metalloproteinases (MMP) constitute a family of more than 25 enzymes which process a large number of pericellular substrates. Even though initially reported to have an ability to degrade almost all of the extracellular components, MMP are now known to play roles which are not limited to the breakdown of extracellular barriers. In fact, MMPs regulate many biological processes, being involved not only in physiological events, but also in pathological processes. Strikingly, MMPs have been found to be involved in the physiology of the Central Nervous System (CNS), taking part and playing important roles in several processes such as repair and ontogeny, as well as in pathological conditions of the CNS. Initially considered to be a static structure, lacking regenerative capability, the CNS has been considered for a long time to be a system without renewal capabilities. Recently, the discovery of constant neural replacement has changed our way of considering the adult brain, and the finding of the existence of neural stem cells has opened the way to exciting and fascinating perspectives of the CNS. So, could MMPs, originally found during metamorphosis in tadpoles, and now amazingly identified in the CNS, have something to do in neuronal function? In this review we take into consideration the possible roles of two metalloproteinases, MMP-2 and MMP-9, also called gelatinases, in controlling several aspects of CNS organization, including the modulation of neural stem cell properties and the differentiation of their progeny, both under normal and pathophysiological conditions.
The International journal of developmental biology 02/2009; 53(1):1-17. · 2.16 Impact Factor
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ABSTRACT: Protein carbonyl levels are the most frequently used biomarker of protein oxidation in several human diseases, including cancer. Breast cancer, a worldwide disease with increasing incidence, develops from ductal/lobular epithelium from which nipple aspirate fluid can be collected and analysed to assess tissue metabolic activity. Our aims were to perform an exploratory investigation on the protein carbonyl accumulation in breast secretions from healthy and cancer patients and its correlation with lipid peroxidation markers.
Protein carbonyls were determined by ELISA in 288 Nipple Aspirate Fluids (NAF) from Control, Pre-malignant and Cancer patients.
Significantly higher protein carbonyl concentration was found in NAF from breast cancer (BC) patients compared to Control subjects. Cancer patients accumulated in NAF significantly higher levels of carbonyls in post-menopausal condition. A significant inverse relationship between carbonyls and 8-F2alpha-isoprostanes in NAF was found in Cancer patients. NAF levels of protein carbonyls are significantly higher in women with pre-malignant conditions than in healthy subjects.
Our results support the hypothesis that oxidative stress in breast microenvironment plays a role in breast cancer; measurement of protein and lipid oxidative products in NAF may improve the identification of women at increased breast cancer risk.
Cellular oncology: the official journal of the International Society for Cellular Oncology 01/2009; 31(5):383-92. · 4.17 Impact Factor
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ABSTRACT: Blood sampling/handling alters matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) expression. The aim of this study is to evaluate the effects of high molecular weight heparin on MMP and TIMP expression in blood.
We analyzed by gelatin zymography and ELISA assays the effects of different heparin salts, dose- and time-dependence of MMP and TIMP concentrations in plasma and sera collected with and without clot-accelerator in plastic tubes from 50 healthy donors.
The levels and zymography of MMP-2 did not show significant changes among all samples, and during time- and dose-dependent heparin treatments. MMP-9 and TIMP-2 expression were strongly affected by heparin, with significant increase of their content and gelatinolytic activity both in time- and in dose-dependent fashion. Addition of heparin allowed also the displacement of MMP-2 prodomain, favouring zymogen activation.
Heparin has direct and indirect effects, altering MMP/TIMP complexes circulating in blood, and increasing the release of TIMP-2. To avoid misinterpretations due to MMP/TIMP complex alteration and MMP prodomain displacement, heparin should be cautiously used in blood collection procedures.
Clinical biochemistry 11/2008; 41(18):1466-73. · 2.02 Impact Factor
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ABSTRACT: Gross cystic breast disease (GCBD) is the most common benign breast disorder, but the molecular basis of cyst formation remains to be identified. If the use of aluminium-based antiperspirant salts is involved in the etiology of gross breast cyst formation, it might be expected that aluminium would be at elevated levels in human breast cyst fluid (BCF). Aluminium was measured by ICP-MS in 48 samples of BCF, 30 samples of human blood serum and 45 samples of human breast milk at different stages of lactation (colostrum, intermediate, mature). The median level of aluminium in apocrine type I BCF (n = 27, 150 microg l(-1)) was significantly higher than in transudative type II BCF (n = 21, 32 microg l(-1); P < 0.0001). By comparison, aluminium measurements gave a median concentration of 6 microg l(-1) in human serum and 25 microg l(-1) in human breast milk, with no difference between colostrum, intermediate and mature milk. Levels of aluminium were significantly higher in both types of BCF than in human serum (P < 0.0001). However when compared with human breast milk, aluminium levels were only significantly higher in apocrine type I BCF (P < 0.0001) and not in transudative type II BCF (P = 0.152). It remains to be identified why such high levels of aluminium were found in the apocrine type I BCF and from where the aluminium originated. However, if aluminium-based antiperspirants are found to be the source and to play any causal role in development of breast cysts, then it might become possible to prevent this common breast disorder.
Journal of Applied Toxicology 10/2008; 29(1):1-6. · 2.48 Impact Factor
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ABSTRACT: The aetiology of breast cancer is complex and multifactorial, and may include diet and xenobiotic compounds. A change in diet affects nutrient levels in blood, but to what extent diet can affect micronutrient concentrations in the breast is not yet well established. Breast nipple aspirate fluids (NAF) can be non-invasively obtained from the breast in most women; it represents a biological tool to assess metabolic changes in the breast ductal microenvironment. A wide variation in biomolecular and hormonal composition of NAFs collected from healthy and breast cancer patient may be due to genetic and nutritional factors; however, micro- and macro-nutrients may influence the secretory status of these women, thus NAF composition and risk of breast carcinoma. The aim of this overview is to highlight the detrimental/beneficial role that diet-related compounds in nipple aspirate fluid can have in breast cancer risk.
Genes & Nutrition 08/2008; 3(2):77-85. · 2.51 Impact Factor
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ABSTRACT: Matrix metalloproteinases (MMPs) are promising diagnostic tools, and blood sampling/handling alters MMP concentrations between plasma and serum and between serum with and without clot activators. To explain the higher MMP-9 expression in serum collected with clot accelerators relative to serum with no additives and to plasma, we analyzed the effects of increasing amounts of silica and silicates (components of clot activators) in citrate plasma, serum, and buffy coats collected in both plastic and glass tubes from 50 healthy donors, and we analyzed the effects of silica and silicate on cultured leukemia cells. The levels of MMP-2 did not show significant changes between glass and plastic tubes, between serum and plasma, between serum with and without clot accelerators, or between silica and silicate treatments. No modification of MMP-9 expression was obtained by the addition of silica or silicate to previously separated plasma and serum. Increasing the amounts of nonsoluble silica and soluble silicate added to citrate and empty tubes prior to blood collection resulted in increasing levels of MMP-9 relative to citrate plasma and serum. Silica and silicate added to buffy coats and leukemia cells significantly induced MMP-9 release/secretion, demonstrating that both silica and silicate induce the release of pro- and complexed MMP-9 forms. We recommend limiting the misuse of serum and avoiding the interfering effects of clot activators.
Analytical Biochemistry 04/2008; 374(1):56-63. · 3.00 Impact Factor
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ABSTRACT: Erythropoietin (Epo) is an important regulator of erythropoiesis, and controls proliferation and differentiation of both erythroid and non-erythroid tissues. Epo is actively synthesized by breast cells during lactation, and also plays a role in breast tissues promoting hypoxia-induced cancer initiation. Our aims are to perform an exploratory investigation on the Epo accumulation in breast secretions from healthy and cancer patients and its localization in breast cancer cells.
Epo was determined by ELISA, immunoprecipitation, western blot and immunocytochemical analyses in 130 Nipple Aspirate Fluids (NAF) from 102 NoCancer and 28 Breast Cancer (BC) patients, comparing results with those found in 10 milk, 45 serum samples and breast cancer cell lines.
Epo levels in NAFs were significantly higher than those in milk and serum. No difference in Epo electrophoretic mobility was found among NAF, milk and serum samples, and conditioned cell culture medium. Immunolocalization of intracellular Epo in ductal cells floating in BC NAFs was similar to those of cancer cell lines. No significant correlation between TNM classification and Epo in NAFs from BC patients was found. Significantly higher Epo concentration was found in NAF from BC patients compared to NoCancer.
We demonstrate that breast epithelial cells are a source of Epo in breast microenvironment, suggesting the presence of a paracrine/autocrine Epo function in NAFs, triggering off intracellular signaling cascade with subsequent BC initiation.
Cellular oncology: the official journal of the International Society for Cellular Oncology 02/2008; 30(1):51-61. · 4.17 Impact Factor
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ABSTRACT: Bone marrow-derived mesenchymal stem cells are a multipotent adult cellular population endowed with broad differentiation potential. Their regeneration capability, ease to undergo gene modifications, and immuno-suppressive capacity makes them optimal tools for tissue engineering, gene- and immuno-therapy. Due to the ever-increasing number of studies on the clinical applications of mesenchymal stem cells in regenerative medicine, these cells have become attractive targets in clinical transplantation. However, the identification and definition of mesenchymal stem cell culture media for their clinical application in cell therapy is currently a matter of strong discussion. Up to now, clinical studies have been conducted with mesenchymal stem cells cultured in foetal calf serum, and the chance of contamination or immunological reaction towards xenogeneic compounds must be taken into consideration. On the other hand, a serum-free medium without the addition of growth factors is not able to expand these cells in vitro; so the evaluation of which is best, among foetal calf serum, human serum (whether autologous or allogeneic) and platelet-rich plasma, is a hot topic urgently needing further research efforts. The need for the establishment of standardized protocols for mesenchymal stem cell preparations, in order not to interfere with their self-renewal and differentiation processes, assuring durable engraftment and long-term therapeutic effects, is evidently crucial. Therefore, the search for optimal culture conditions for the effective clinical-scale production of vast numbers of mesenchymal stem cells for cellular therapy is of paramount importance and the need for a robust passage from basic to translational research is fundamental.
The International Journal of Developmental Biology 02/2008; 52(8):1023-32. · 2.82 Impact Factor
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Clinical Chemistry 12/2007; 53(11):1981-2. · 7.91 Impact Factor
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ABSTRACT: The matrix metalloproteinases (MMPs), responsible for the degradation of extracellular matrix (ECM) proteins, may regulate brain cellular functions. Choline acetyltransferase (ChAT) transfected murine neuroblastoma cell line N18TG2, that synthesize acetylcholine and show enhancement of several neurospecific markers (i.e., sinapsin I, voltage gated Na(+) channels, high affinity choline uptake) and fiber outgrowth, were studied for the MMP regulation during neuronal differentiation. Zymography of N18TG2 culture medium revealed no gelatinolytic activity, whereas after carbachol treatment of cells both MMP-9 and activated MMP-2 forms were detected. ChAT-transfected clone culture medium contains three MMP forms at 230, 92, and 66kDa. Carbachol treatment increased MMP-2 and MMP-9 gene expression in N18TG2 cells and higher levels for both genes were also observed in ChAT transfected cells. The data are consistent with the hypothesis that acetylcholine brings about the activation of an autocrine loop modulating MMP expression.
Biochemical and Biophysical Research Communications 11/2007; 362(2):269-74. · 2.48 Impact Factor
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International Journal of Cancer 08/2007; 121(1):216-8; author reply 219-23. · 5.44 Impact Factor
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ABSTRACT: The choice of an optimal strategy of stem cell culture is at the moment an impossible task, and the elaboration of a culture medium adapted to the production of embryonic and adult mesenchymal stem cells for the clinical application of cell therapy remains a crucial matter. To make an informed choice, it is crucial to not underestimate the theoretical health risk of using xenogenic compounds, to limit the immunological reactions once stem cells are transplanted, to not overestimate the controversial results obtained with human serum, plasma, and blood derivatives, as well as to carefully examine the pros and cons of serum-free and ad hoc formulation strategies; besides that, to also maintain multipotentiality, self-renewal, and transplantability. The extent to which we are able to achieve effective cell therapies will depend on assimilating a rapidly developing base of scientific knowledge with the practical considerations of design, delivery, and host response. Although clinical studies have already started, many questions remain unsolved, and concomitantly even more evidence on suitable and safe off-the-shelf products (mainly xeno-free) for embryonic and mesenchymal stem cells is cropping up, even though there should be no rush to enter the clinical stage while the underlying basic research is still not so solid; this solely will lead to high-quality translational research, without making blunders stemming from the assumption that all that glitters is not gold. Disclosure of potential conflicts of interest is found at the end of this article.
Stem Cells 07/2007; 25(7):1603-9. · 7.78 Impact Factor
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ABSTRACT: The choice of an optimal strategy of stem cell culture is at the moment an impossible task, and the elaboration of a culture medium adapted to the production of embryonic and adult mesenchymal stem cells for the clinical application of cell therapy remains a crucial matter. To make an informed choice, it is crucial to not underestimate the theoretical health risk of using xenogenic compounds, to limit the immunological reactions once stem cells are transplanted, to not overestimate the controversial results obtained with human serum, plasma, and blood derivatives, as well as to carefully examine the pros and cons of serum-free and ad hoc formulation strategies; besides that, to also maintain multipotentiality, self-renewal, and transplantability. The extent to which we are able to achieve effective cell therapies will depend on assimilating a rapidly developing base of scientific knowledge with the practical considerations of design, delivery, and host response. Although clinical studies have already started, many questions remain unsolved, and concomitantly even more evidence on suitable and safe off-the-shelf products (mainly xeno-free) for embryonic and mesenchymal stem cells is cropping up, even though there should be no rush to enter the clinical stage while the underlying basic research is still not so solid; this solely will lead to high-quality translational research, without making blunders stemming from the assumption that all that glitters is not gold.Disclosure of potential conflicts of interest is found at the end of this article.
Stem Cells 06/2007; 25(7):1603 - 1609. · 7.78 Impact Factor
