Hypoxia and neural stem cells: from invertebrates to brain cancer stem cells.

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.

Analysis of aluminium content and iron homeostasis in nipple aspirate fluids from healthy women and breast cancer-affected patients.

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.

Iron-binding proteins and C-reactive protein in Nipple Aspirate Fluids: role of Iron-driven inflammation in breast cancer microenvironment?

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.

Detection of superoxide dismutase-1 in nipple aspirate fluids: a reactive oxygen species-regulating enzyme in the breast cancer microenvironment.

INTRODUCTION: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Protein oxidation in breast microenvironment: Nipple aspirate fluid collected from breast cancer women contains increased protein carbonyl concentration.

BACKGROUND: 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. METHODS: Protein carbonyls were determined by ELISA in 288 Nipple Aspirate Fluids (NAF) from Control, Pre-malignant and Cancer patients. RESULTS: 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. CONCLUSION: 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.

Protein profile analysis of the breast microenvironment to differentiate healthy women from breast cancer patients.

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.

Statins and breast cancer: may matrix metalloproteinase be the missing link.

Statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors), cholesterol-lowering agents widely prescribed for cardiovascular health, have been shown to exert several pleiotropic effects. Although some studies reported that statins have no effects on malignancies of any kind, results of several epidemiologic and in vitro studies highlighted that statins exert anticancer activity in various cell types, showing that long-term therapy inhibits the incidence and/or progression of some human tumours. In particular, in the present overview we focused the attention on a neglected aspect of the pleiotropic functions of some lipophilic statins, suggesting that the possible mechanism of matrix metalloproteinase downregulation arises from prolonged lowering of circulating cholesterol. Our hypothesis may explain the literary findings about the phenomenon of switching of breast cancer phenotypes by statins, shedding the basis of future epidemiologic and basic science studies about the role of circulating and/or tumor-resident cholesterol in the initiation and progression of breast cancer.

Neural stem cells at the crossroads: MMPs may tell the way.

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.

Concentration of aluminium in breast cyst fluids collected from women affected by gross cystic breast disease.

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.

Intracrinology of breast microenvironment: hormonal status in nipple aspirate fluid and its relationship to breast cancer.

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.

Nutrients and nipple aspirate fluid composition: the breast microenvironment regulates protein expression and cancer aetiology.

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.

Heparin affects matrix metalloproteinases and tissue inhibitors of metalloproteinases circulating in peripheral blood.

OBJECTIVES: 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. DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

From bone marrow to therapeutic applications: different behaviour and genetic/epigenetic stability during mesenchymal stem cell expansion in autologous and foetal bovine sera?

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.

Increased shedding of soluble fragments of P-cadherin in nipple aspirate fluids from women with breast cancer.

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 breast metabolic activity. P-cadherin is frequently over-expressed in high-grade invasive breast carcinomas and has been reported to be an enhancer of migration and invasion of breast cancer cells, being correlated with tumor aggressiveness. The present study analyzed the soluble fragment of P-cadherin in milk, NAF and matched plasma samples of healthy subjects and in women with precancer conditions and breast cancer. Soluble P-cadherin was detected in all plasma and milk samples, and in about 31.3% of NAF samples. The lowest levels of soluble P-cadherin were found in plasma, with no significant difference among NoCancer, PreCancer and Cancer patients. The highest concentration of soluble P-cadherin was detected in milk collected during the first trimester of lactation, significantly with respect to all NAF samples. There were significantly higher levels of soluble P-cadherin in NAF from Cancer patients than those in women with NoCancer and PreCancer (P < 0.0001). Although no significant difference was found between in situ and invasive breast cancer, soluble P-cadherin levels were found at high concentrations in c-erbB-2-positive tumors, showing a positive correlation with disease stage grouping and tumor grade, and an inverse relationship with estrogen/progesterone receptor status. High levels of the soluble fragment of P-cadherin in Cancer NAF suggest its possible release via proteolytic processing, favoring cancer cell detachment from breast duct, and suggesting that measuring soluble P-cadherin in NAF may improve the identification of women with increased breast cancer risk.

Erythropoietin and its receptor in breast cancer: putting together the pieces of the puzzle.

The expression of erythropoietin (Epo) and the Epo receptor (EpoR) has been detected in healthy tissue as well as in a variety of human cancers, including breast. Functional Epo/EpoR signaling in cancer cells, which contributes to disease initiation/progression, is not completely straightforward and is difficult to reconcile with the clinical practice of preventing/treating anemia in cancer patients with recombinant Epo. Preclinical and clinical investigations have provided contrasting results, ranging from a beneficial role that improves the patient's overall survival to a negative impact that promotes tumor growth progression. A careful gathering of Epo/EpoR biomolecular information enabled us to assemble an unexpected jigsaw puzzle which, via distinct JAK-dependent and JAK-independent mechanisms and different internalization/recycling as well as ubiquitination/degradation pathways, could explain most of the controversies of preclinical and clinical studies. However, until the mechanisms of the contrasting literature data are resolved, this new point of view may shed light on the Epo/EpoR paracrine/autocrine system and function, providing a basis for further studies in order to achieve the highest possible benefit for cancer patients.

Increased levels of erythropoietin in nipple aspirate fluid and in ductal cells from breast cancer patients.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Differences in both matrix metalloproteinase 9 concentration and zymographic profile between plasma and serum with clot activators are due to the presence of amorphous silica or silicate salts in blood collection devices.

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.

Nipple aspirate fluids from women with breast cancer contain increased levels of group IIa secretory phospholipase A2.

Arachidonic acid, a bioactive molecule metabolized into prostaglandins and leukotrienes, contributes to cellular proliferation and tumor progression. Group IIa secretory phospholipase A2 (sPLA2-IIa) can facilitate arachidonate release from cellular phospholipids, suggesting its involvement in tumor evolution. Analysis of breast nipple aspirate fluid (NAF), a noninvasive research tool, allows the identification of biomarkers of breast cancer. We sought to determine whether sPLA2-IIa expression might be related to breast cancer development or progression. sPLA2-IIa expression was evaluated in NAF samples from 110 women (57 women with and 53 without breast cancer) using ELISA and western blotting; ultrastructural immunolocalization was performed in epithelial cells floating in NAF. Immunocytochemistry revealed that sPLA2-IIa is a constitutive intracellular protein suggesting that breast ductal cells synthesize and secrete the 14 kDa protein in NAF. Among all 110 subjects, sPLA2-IIa expression was significantly increased both in NAF and within ductal epithelial cells from cancer containing breasts. While in healthy women menopausal status did not influence sPLA2-IIa expression (P = 0.457), among patients with breast cancer there was a significant down-regulation in postmenopausal subjects (P < 0.0001). Moreover, sPLA2-IIa concentration in NAF from breast cancer patients was positively correlated with tumor stage (r (2) = 0.979, P = 0.0012), suggesting an active secretion/accumulation of the enzyme in NAF based on tumor burden. sPLA2-IIa activity may serve a dual role in breast carcinogenesis, beneficial in its release of arachidonate and detrimental in the metabolic conversion of arachadonic acid into prostaglandins and leukotrienes.

Acetylcholine induces neurite outgrowth and modulates matrix metalloproteinase 2 and 9.

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.