Nanoparticle-biomolecular corona: A new approach for the early detection of non-small-cell lung cancer.

Lung cancer (LC) is the most common type of cancer and the second cause of death worldwide in men and women after cardiovascular diseases. Non-small-cell lung cancer (NSCLC) is the most frequent type of LC occurring in 85% of cases. Developing new methods for early detection of NSCLC could substantially increase the chances of survival and, therefore, is an urgent task for current research. Nowadays, explosion in nanotechnology offers unprecedented opportunities for therapeutics and diagnosis applications. In this context, exploiting the bio-nano-interactions between nanoparticles (NPs) and biological fluids is an emerging field of research. Upon contact with biofluids, NPs are covered by a biomolecular coating referred to as "biomolecular corona" (BC). In this study, we exploited BC for discriminating between NSCLC patients and healthy volunteers. Blood samples from 10 NSCLC patients and 5 subjects without malignancy were allowed to interact with negatively charged lipid NPs, leading to the formation of a BC at the NP surface. After isolation, BCs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We found that the BCs of NSCLC patients was significantly different from that of healthy individuals. Statistical analysis of SDS-PAGE results allowed discriminating between NSCLC cancer patients and healthy subjects with 80% specificity, 80% sensitivity and a total discriminate correctness rate of 80%. While the results of the present investigation cannot be conclusive due to the small size of the data set, we have shown that exploitation of the BC is a promising approach for the early diagnosis of NSCLC.

The enigmatic role of matrix metalloproteinases in epithelial-to-mesenchymal transition of oral squamous cell carcinoma: Implications and nutraceutical aspects.

The most prevalent malignancy in the oral cavity is represented by oral squamous cell carcinoma, an aggressive disease mostly detected in low-income communities. This neoplasia is mostly diffused in older men particularly exposed to risk factors such as tobacco, alcohol, and a diet rich in fatty foods and poor in vegetables. In oral squamous cell carcinoma, a wide range of matrix-cleaving proteinases are involved in extracellular matrix remodeling of cancer microenvironment. In particular, matrix metalloproteinases (MMPs) represent the major and most investigated protagonists. Owing to their strong involvement in malignant pathologies, MMPs are considered the most promising new biomarkers in cancer diagnosis and prognosis. The interest in studying MMPs in oral cancer biology is also owing to their prominent role in epithelial-to-mesenchymal transition (EMT). EMT is an intricate process involving different complex pathways. EMT-related proteins are attractive diagnostic biomarkers that characterize the activation of biological events that promote cancer's aggressive expansion. Different antioncogenic natural compounds have been investigated to counteract oral carcinogenesis, with the scope of obtaining better clinical results and lower morbidity. In particular, we describe the role of different nutraceuticals used for the regulation of MMP-related invasion and proliferation of oral cancer cells.

Multifaceted Breast Cancer: The Molecular Connection With Obesity.

Obesity is characterized by a disruption in energy balance regulation that results in an excess accumulation of body fat. Its increasing prevalence poses a major public health concern because it is a risk factor for a host of additional chronic conditions, including type 2 diabetes, hypertension, and cardiovascular disease. Obesity is increasingly recognized as a growing cause of cancer risk. In particular excessive adipose expansion during obesity causes adipose dysfunction and inflammation that can regulate tumor growth. In obesity, dysregulated systemic metabolism and inflammation induce hyperinsulinemia, hyperglycemia, dyslipidemia, and enhance sex hormone production with increased secretion of proinflammatory adipokine that impact breast cancer development and progression. This review describes how adipose inflammation that characterizes obesity is responsible of microenvironment to promote cancer, and discuss how steroid hormones, that are essential for the maintenance of the normal development, growth and differentiation of the cells, influence the induction and progression of breast cancer. J. Cell. Physiol. 232: 69-77, 2017. © 2016 Wiley Periodicals, Inc.

Evaluation of matrix metalloproteinase type IV-collagenases in serum of patients with tumors of the central nervous system.

The basement membrane collagen IV-degrading matrix metalloproteinases -2 and -9 (MMPs) are most often linked to the malignant phenotype of tumor cells by playing a critical role in invasion, metastasis, angiogenesis, and vasculogenesis. We verified the activity of these two MMPs in the sera of patients affected by brain tumors (20 gliomas, 28 meningiomas and 20 metastasis) by zymography. The sera of 25 healthy volunteers with no concomitant illnesses were used for controls. Zymography showed four dominant gelatinolytic bands of 240, 130, 92 (MMP-9) and 72 (MMP-2) kDa. No statistically significant variations of MMP-2 proteolytic activity between patients and healthy individuals were observed. On the contrary, MMP-9 (both monomeric and multimeric forms) lytic activities were significantly higher in tumors specimens compared to healthy controls (p < 0.001). Moreover, MMP-9 immunohistochemistry revealed: (1) a strong reactivity in neoplastic vessels of high-grade gliomas showing an inverse correlation with serum multimeric gelatinolytic activity; (2) a cytoplasmatic reactivity in meningiomas with a significantly increase in atypical meningioma compared with low-grade ones (p = 0.036); (3) a positive correlation between MMP-9 and Ki-67 (Sperman Rho coefficient r = 0.418 and p = 0.034). Our results suggest that serum and tissue MMP-9 might provide clinicians additional objective information in intracranial neoplasms. Finally, it should be possible to use MMP-9 as a target for new forms of therapy. Nevertheless, due to the small number of patients included in the study, the conclusion may not be transferable to the general population and therefore further evaluations are needed.

Translational Research and Plasma Proteomic in Cancer.

Proteomics is a recent field of research in molecular biology that can help in the fight against cancer through the search for biomarkers that can detect this disease in the early stages of its development. Proteomic is a speedily growing technology, also thanks to the development of even more sensitive and fast mass spectrometry analysis. Although this technique is the most widespread for the discovery of new cancer biomarkers, it still suffers of a poor sensitivity and insufficient reproducibility, essentially due to the tumor heterogeneity. Common technical shortcomings include limitations in the sensitivity of detecting low abundant biomarkers and possible systematic biases in the observed data. Current research attempts are trying to develop high-resolution proteomic instrumentation for high-throughput monitoring of protein changes that occur in cancer. In this review, we describe the basic features of the proteomic tools which have proven to be useful in cancer research, showing their advantages and disadvantages. The application of these proteomic tools could provide early biomarkers detection in various cancer types and could improve the understanding the mechanisms of tumor growth and dissemination.

Substrate-zymography: a still worthwhile method for gelatinases analysis in biological samples.

Matrix metallo-proteinases (MMPs) are a family of zinc-dependent endopeptidases, capable of degrading all the molecular components of extracellular matrix. A class of MMPs is gelatinases which includes gelatinase A or MMP-2 (72 kDa) and gelatinase B or MMP-9 (92 kDa), which have been shown to play critical roles in pathophysiology of many human disease and, in particular, cancer progression. For these reasons they obtained a great interest as potential non-invasive biomarker in providing useful clinical information in cancer diagnosis and therapy. A sensitive and unexpensive method for analysis of gelatinases is the gelatine zymography, which allows to measure the relative amounts of active and inactive enzymes in body fluids and tissue extracts. The procedure involves the electrophoretic separation of proteins under denaturing but non reducing conditions through a polyacrylamide gel containing a synthetic substrate (gelatin). The aim of this mini-review has been to describe the general principles of gelatine zymography technique, underling the main advantages and disadvantages. Even though an improvement of this method is necessary for a better applicability in laboratory medicine, gelatine zymography represents the most convenient method to detect the activity of the different gelatinases from a wide range of biological samples.

Oncogenic MicroRNAs Characterization in Clear Cell Renal Cell Carcinoma.

A key challenge for the improvement of clear cell renal cell carcinoma (ccRCC) management could derive from a deeper characterization of the biology of these neoplasms that could greatly improve the diagnosis, prognosis and treatment choice. The aim of this study was to identify specific miRNAs that are deregulated in tumor vs. normal kidney tissues and that could impact on the biology of ccRCC. To this end we selected four miRNAs (miR-21-5p, miR-210-3p, miR-185-5p and miR-221-3p) and their expression has been evaluated in a retrospective cohort of formalin-fixed paraffin-embedded (FFPE) tissues from 20 ccRCC patients who underwent surgical nephrectomy resection. miR-21-5p and miR-210-3p resulted the most significantly up-regulated miRNAs in this patient cohort, highlighting these onco-miRNAs as possible relevant players involved in ccRCC tumorigenesis. Thus, this study reports the identification of specific oncogenic miRNAs that are altered in ccRCC tissues and suggests that they might be useful biomarkers in ccRCC management.

Preoperative insulin-like growth factor-binding protein-3 (IGFBP-3) blood level predicts gleason sum upgrading.

BACKGROUND: About 43% of men with low Gleason grade prostate cancer (PCa) at biopsy will be finally diagnosed with high-grade PCa at radical prostatectomy (RP). Gleason sum at RP is a good indicator of biochemical recurrence and poor clinical outcome. Therefore, there is a need to improve clinical evaluation of PCa aggressiveness in order to choice appropriate treatment. To this aim an easy-available tool is represented by circulating biomarkers. Among these, the best candidates are some molecules involved in PCa pathogenesis such as IGFBP-2 and IGFBP-3, IL-6, and its soluble receptor (SIL-6R). METHODS: In this study, we evaluated the ability of preoperative IGFBP-2, IGFBP-3, IL-6, and SIL-6R serum levels to predict Gleason score upgrade in 52 PCa patients. RESULTS: We found that IGFBP-3 median levels were significantly lower in patients who showed Gleason upgrading from biopsy to RP (P = 0.024). We also found an association between biopsy T-stage and Gleason Upgrade (P = 0.011). Using multivariate logistic regression model, we demonstrated that the association of IGFBP-3 serum levels together with biopsy T-stage and biopsy Gleason score was useful to calculate a prognostic risk score. ROC curve analysis of risk score showed a good ability to predict GSU (AUC = 0.81; 95% CI 0.69-0.93). CONCLUSIONS: Our results suggest that preoperative IGFBP-3 circulating levels determination may be useful to predict Gleason score upgrading alone and/or in combination with biopsy T-stage and biopsy Gleason score.

Expression of Beta-Catenin, Cadherins and P-Runx2 in Fibro-Osseous Lesions of the Jaw: Tissue Microarray Study.

Fibrous dysplasia (FD) and hyperparathyroidism-jaw tumor syndrome (HPT-JT) are well-characterized benign bone fibro-osseous lesions. The intracellular mechanism leading to excessive deposition of fibrous tissue and alteration of differentiation processes leading to osteomalacia have not yet been fully clarified. Tissue Microarray (TMA)-based immunohistochemical expression of ß-catenin, CK-AE1/AE3, Ki-67, cadherins and P-Runx2 were analyzed in archival samples from nine patients affected by FD and HPT-JT and in seven controls, with the aim of elucidating the contribution of these molecules (ß-catenin, cadherins and P-Runx2) in the osteoblast differentiation pathway. ß-catenin was strongly upregulated in FD, showing a hyper-cellulated pattern, while it was faintly expressed in bone tumors associated with HPT-JT. Furthermore, the loss of expression of OB-cadherin in osteoblast lineage in FD was accompanied by N-cadherin and P-cadherin upregulation (p < 0.05), while E-cadherin showed a minor role in these pathological processes. P-Runx2 showed over-expression in six out of eight cases of FD and stained moderately positive in the rimming lining osteoblasts in HPT-JT syndrome. ß-catenin plays a central role in fibrous tissue proliferation and accompanies the lack of differentiation of osteoblast precursors in mature osteoblasts in FD. The study showed that the combined evaluation of the histological characteristics and the histochemical and immunohistochemical profile of key molecules involved in osteoblast differentiation are useful in the diagnosis, classification and therapeutic management of fibrous-osseous lesions.

Protein corona profile of graphene oxide allows detection of glioblastoma multiforme using a simple one-dimensional gel electrophoresis technique: a proof-of-concept study.

Glioblastoma multiforme (GBM) is the most aggressive form of gliomas. The development of supplementary approaches for glioblastoma diagnosis, limited to imaging techniques and tissue biopsies so far, is a necessity of clinical relevance. In this context, nanotechnology might afford tools to enable early diagnosis. Upon exposure to biological media, nanoparticles are coated with a layer of proteins, the protein corona (PC), whose composition is individual and personalized. Here we show that the PC of graphene oxide nanosheets has a capacity to detect GBM using a simple one-dimensional gel electrophoresis technique. In a range of molecular weights between 100 and 120 kDa, the personalized PC from GBM patients is completely discernible from that of healthy donors and that of cancer patients affected by pancreatic adenocarcinoma and colorectal cancer. Using tandem mass spectrometry, we found that inter-alpha-trypsin inhibitor (ITI) heavy chain H4 is enriched in the PC of all tested individuals but not in the GBM patients. Overall, if confirmed on a larger cohort series, this approach could be advantageous at the first level of investigation to decide whether to carry out more invasive analyses and/or to follow up patients after surgery and/or pharmacological treatment.

Sex Hormones and Inflammation Role in Oral Cancer Progression: A Molecular and Biological Point of View.

Oral cancers have been proven to arise from precursors lesions and to be related to risk behaviour such as alcohol consumption and smoke. However, the present paper focuses on the role of chronic inflammation, related to chronical oral infections and/or altered immune responses occurring during dysimmune and autoimmune diseases, in the oral cancerogenesis. Particularly, oral candidiasis and periodontal diseases introduce a vicious circle of nonhealing and perpetuation of the inflammatory processes, thus leading toward cancer occurrence via local and systemic inflammatory modulators and via genetic and epigenetic factors.

Expression and clinical implication of cyclooxygenase-2 and E-cadherin in oral squamous cell carcinomas.

Epithelial-Mesenchymal Transition (EMT) and angiogenesis are crucial events for development of aggressive and often fatal Oral Squamous Cell Carcinomas (OSCCs). Both promote cancer progression and metastasis development, but while the former induces the loss of E-cadherin expression and, hence cadherin switching; the latter produces hematic blood vessel neo-formation and contribute to OSCC cell growth, tumor mass development, and dissemination. Cyclooxygenase-2 (COX-2) has an important role, not only in angiogenic mechanisms, but also in favoring cancer invasion. Indeed it decreases the expression of E-cadherin and leads to phenotypic changes in epithelial cells (EMT) enhancing their carcinogenic potential. Our aim is to evaluate the interplay between E-cadherin cytoplasmic delocalization, COX-2 up-regulation and COX-2 induced neo-angiogenesis in 120 cases of OSCC. We have analyzed the distribution and the number of neo-formed endothelial buds surrounding infiltrating cells that express COX-2, as well as the neo-formed vessels in chronic inflammatory infiltrate, which surround the tumor. A double immunostaining method was employed in order to verify co-localization of endothelial cell marker (CD34) and COX-2. IHC has also been used to assess E-cadherin expression. Our data demonstrate that the OSCC cells, which lose membranous E-cadherin staining, acquiring a cytoplasmic delocalization, overexpress COX-2. Moreover, we find a new CD34+ vessel formation (sprouting angiogenesis). Only basaloid type of OSCC showes low level of COX-2 expression together with very low level of neo-angiogenesis and consequent tumor necrosis. The well-known anti-metastatic effect of certain COX-2 inhibitors suggests that these molecules might have clinical utility in the management of advanced cancers.

Emerging role of secreted miR-210-3p as potential biomarker for clear cell Renal Cell Carcinoma metastasis.

BACKGROUND: MicroRNAs (miRNAs) are emerging as promising molecules in the diagnosis, prognosis and treatment of urological tumours. Recently, our group performed two independent studies highlighting that miR-210-3p may be a useful biomarker not only for diagnosis but also for post-surgery clear cell Renal Cell Carcinoma (ccRCC) management. OBJECTIVE: The aim of this study is to further explore the effectiveness of miRNA as non-invasive biomarker for clinical outcomes and ccRCC response to the treatment. METHODS: We analyzed miR-210-3p levels in neoplastic and healthy tissue and in urine specimens collected at surgery and during follow-up of 21 ccRCC patients by RTqPCR. RESULTS: Firstly, we confirmed that the expression of miR-210-3p was upregulated in tumor tissues and in urine samples of analyzed cohort. Of note is that miR-210-3p expression was significantly reduced in urine samples from disease-free patients during follow-up (from 3 to 12 months) compared to the baseline levels observed at the time of surgery. In a small subgroup of patients presenting metastatic progression (such as bone, intestinal or lung metastasis), the urine levels of miR-210-3p correlated with responsiveness to the therapy. CONCLUSIONS: This pilot study highlights the relevance of secreted miR-210-3p as powerful non-invasive prognostic and predictive biomarker for the evaluation of clinical outcomes and treatment response during ccRCC follow up.

Prep1 Deficiency Affects Olfactory Perception and Feeding Behavior by Impairing BDNF-TrkB Mediated Neurotrophic Signaling.

Prep1 is a homeodomain transcription factor which has an important role in hindbrain development. Prep1 expression is also kept in adult mouse brain and in particular within the olfactory bulbs. Moreover, many Prep1 neurons co-localize with Calbindin-positive periglomerular interneurons in olfactory glomerular layer. However, Prep1 function in this brain region is still unknown. In this study, we show that Prep1 hypomorphic heterozygous (Prep1i/+) mice express low levels of protein and feature a 30% reduction of olfactory bulb area, compared to WT mice. In addition, Prep1i/+ mice olfactory bulb histological analysis indicated a 20% lower cytochrome C oxidase activity within the glomerular layer, accompanied by a reduced number of periglomerular interneurons, compared to the WT littermates. Consistently, olfactory perception test highlighted that Prep1 hypomorphic heterozygous mice display a scant ability to distinguish odors, which significantly impacts on feeding behavior, as Prep1i/+ mice revealed a reduced preference for high-fat food. Analysis of BDNF signaling, which represents the main molecular mediator of olfactory plasticity, showed that Prep1i/+ mouse olfactory bulbs feature a 30% reduction of TrkB receptor levels and a decreased activation of ERK1/2. Similarly, overexpression of Prep1 in mouse neuronal cells (N2A) caused an increase of TrkB expression levels, BDNF-induced ERK phosphorylation, and cell viability, compared to control cells. We conclude that Prep1 deficiency alters olfactory morpho-functional integrity and olfaction-mediated eating behavior by affecting BDNF-TrkB signaling. Prep1 could, therefore, play a crucial role in behavioral dysfunctions associated to impaired responsiveness to BDNF.

Gelatinolytic activities (matrix metalloproteinase-2 and -9) and soluble extracellular domain of Her-2/neu in pleural effusions.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that are implicated in multiple stages of cancer progression including invasion and metastasis. MMPs exert these effects by cleaving a diverse group of substrates, which include not only structural components of the extracellular matrix, but also growth factor receptors. By gelatin zymography we verified MMP activity in the pleural effusions of patients with benign and malignant disease. Of these patients, 32 had malignant pleural effusion, consisting of 20 breast cancer, 6 non-small cell lung carcinoma, 4 ovarian carcinoma, and 2 colonic adenocarcinoma, and 10 had benign pleural effusion (5 pleurisy and 5 cirrhosis). Zymography showed the constant presence of a substantial amount of MMP-2 in all samples analyzed, whereas MMP-9 was present to lesser quantities. MMP-2 activity was enhanced in pleural effusions from patients with benign diseases compared with cancer patients. MMP-9 was present in 59% of cancer patients and the lytic activity was enhanced in pleurisy and absent in cirrhosis. Furthermore, we determined the pleural effusion levels of the soluble extracellular domain of HER-2/neu. The levels of HER-2/neu ECD were above the cut-off value in breast cancer patients. No correlation between gelatinolytic activities and high HER-2/neu ECD values was found.

Secreted miR-210-3p as non-invasive biomarker in clear cell renal cell carcinoma.

The most common subtype of renal cell carcinoma (RCC) is clear cell RCC (ccRCC). It accounts for 70-80% of all renal malignancies representing the third most common urological cancer after prostate and bladder cancer. The identification of non-invasive biomarkers for the diagnosis and responsiveness to therapy of ccRCC may represent a relevant step-forward in ccRCC management. The aim of this study is to evaluate whether specific miRNAs deregulated in ccRCC tissues present altered levels also in urine specimens. To this end we first assessed that miR-21-5p, miR-210-3p and miR-221-3p resulted upregulated in ccRCC fresh frozen tissues compared to matched normal counterparts. Next, we evidenced that miR-210-3p resulted significantly up-regulated in 38 urine specimens collected from two independent cohorts of ccRCC patients at the time of surgery compared to healthy donors samples. Of note, miR-210-3p levels resulted significantly reduced in follow-up samples. These results point to miR-210-3p as a potential non-invasive biomarker useful not only for diagnosis but also for the assessment of complete resection or response to treatment in ccRCC management.

Urinary gelatinase activities (matrix metalloproteinases 2 and 9) in human bladder tumors.

The ability to degrade type IV collagen, the major component of the basement membrane, is unique to gelatinases A and B. These two matrix metalloproteinases (MMPs) are most often linked to the malignant phenotype of tumor cells, and their expression is elevated in several cases of human tumor aggressiveness and overall survival. By gelatin zymography, we verified MMP activity in the urine of patients with bladder cancer. Of these patients, 10 had well-, 8 had moderately and 7 had poorly differentiated bladder cancer. The urine of healthy volunteers with no evidence of disease was used for controls. Zymography showed five dominant gelatinolytic bands of 240, 220, 130, 92 and 72 kDa in tumor samples, whereas only traces of MMP were detected in the urine of healthy subjects. The majority of cancerous urine samples showed MMP-9 lytic activity but only a few contained MMP-2. Moreover, MMP-9 content is enhanced in the urine from patients with high-grade and advanced-stage bladder tumors. Finally, we determined the urinary levels of urinary bladder cancer (UBC), tissue polypeptide-specific antigen (TPS) and protein 22 of nuclear matrix (NMP22). The levels of TPS and NMP-22 were higher in G3 bladder cancer than in G1 and G2 neoplasias. The urinary values of these two biomarkers correlated with the increase in MMP-9 lytic activity in high-grade and advanced-stage bladder cancer.

Malvidin's Effects on Rat Pial Microvascular Permeability Changes Due to Hypoperfusion and Reperfusion Injury.

The present study was aimed to evaluate the malvidin's protective effects on damage induced by 30 min bilateral common carotid artery occlusion (BCCAO) and 60 min reperfusion (RE) in rat pial microcirculation. Rat pial microcirculation was observed using fluorescence microscopy through a closed cranial window. Western blotting analysis was performed to investigate the endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS) and matrix metalloproteinase 9 (MMP-9) expression. Moreover, MMP-9 activity was evaluated by zymography. Finally, neuronal damage and radical oxygen species (ROS) formation were assessed. In all animals, pial arterioles were classified in five orders of branching according to Strahler's method. In hypoperfused rats, 30 min BCCAO and 60 min RE caused a decrease in arteriolar diameter, an increase in microvascular leakage and leukocyte adhesion, accompanied by decreased capillary perfusion and red blood cell velocity (VRBC). Moreover, marked neuronal damage and evident ROS generation were detected. Conversely, malvidin administration induced arteriolar dilation in dose-related manner, reducing microvascular leakage as well as leukocyte adhesion. Capillary perfusion and VRBC were protected. Nitric oxide (NO) synthase inhibition significantly attenuated malvidin's effects on arteriolar diameter. Western blotting analysis revealed an increase in eNOS and p-eNOS expression, while zymography indicated a decrease in MMP-9 activity after malvidin's administration. Furthermore, malvidin was able to prevent neuronal damage and to decrease ROS generation. In conclusion, malvidin protects rat pial microcirculation against BCCAO/RE injury, preventing blood-brain impairment and neuronal loss. Malvidin's effects appear to be mediated by eNOS activation and scavenger activity.

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in diagnosis of pleural effusion of malignant origin.

OBJECTIVES: The aim of the present study was to evaluate the diagnostic accuracy of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in differentiating benign from malignant exudative pleural effusions. METHODS: This is a unicentre observational study including 97 consecutive patients with exudative pleural effusions. Metalloproteinase-9, tissue inhibitor of metalloproteinase-1, lactate dehydrogenase, ferritin, carcinoembryonic antigen and carbohydrate antigen 15-3 were measured in pleural effusion and serum by enzyme-linked immunosorbent assay. The activity of metalloproteinase-9 was also evaluated by substrate zymography. The data were correlated with final diagnosis of pleural effusions to evaluate the diagnostic accuracy. RESULTS: Of the 97 eligible patients, 6 were excluded. Of the 91 patients included in the study, 70 had malignant pleural effusions and 21 had benign pleural effusions. Both in sera and pleural effusions, matrix metalloproteinase-9 (P < 0.0001), tissue inhibitor of metalloproteinase-1 (P < 0.0001) and carcinoembryonic antigen (P < 0.0001) levels were higher in neoplastic patients than in benign group. Zymography analysis showed a most prominent band at a molecular weight of 92 kDa (metalloproteinase-9) whereas a less intense band was observed at 72 kDa (metalloproteinase-2). A significant correlation was found between metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels in pleural effusion (P < 0.0001; r = 0.8) and serum (P < 0.03; r = 0.2). Pleural effusion metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels showed higher value of sensitivity (97 and 91%, respectively) and specificity (90 and 95%, respectively) compared with other standard markers. Serum metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels showed similar results. Among 70 neoplastic patients, 29 had negative pleural cytology. Of these, 25 presented elevated levels of metalloproteinase-9 and tissue inhibitor of metalloproteinase-1, whereas 4 patients had elevated levels of one of the two markers. CONCLUSIONS: Our results showed that metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 might be valuable markers in differentiating benign from malignant pleural effusions. Their levels are neither influenced by the histology and tumour origin nor by the presence of tumour cells in pleural effusions. Thus, their use in clinical practice could help in the selection of patients needing more invasive procedures, such as thoracoscopic biopsy.

Matrix metalloproteinase-2 and matrix metalloproteinase-9 type IV collagenases in serum of patients with pleural effusions.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that are essentially involved in the turnover of the extracellular matrix (ECM). Recently, several MMPs were implicated in creating an environment that supports the initiation and maintenance of tumor growth. We verified MMP activity in the sera of patients with pleural effusions by gelatin zymography. Of these patients, 22 had malignant pleural effusion, consisting of 11 breast carcinomas and 11 lung carcinomas (7 squamous cell carcinomas and 4 adenocarcinomas), and 8 patients had benign effusions. The sera of 25 healthy subjects were used as controls. Zymography analysis showed three major gelatinolytic bands of 72, 92 and 220 kDa. The MMP-9/MMP-2 ratio was enhanced in cancer patients compared with benign diseases and healthy individuals. Furthermore, we determined the circulating levels of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (Ca 19-9), carbohydrate antigen 15-3 (Ca 15-3). The serum levels of Ca 15-3 were above the cut-off levels in several cancer patients. No correlation between gelatinolytic activity and high tumoral marker values was found.