Lactobacillus rhamnosus GG cell-free supernatant as a novel anti-cancer adjuvant.

BACKGROUND: Gut microbiota modulation has been demonstrated to be effective in protecting patients against detrimental effects of anti-cancer therapies, as well as to improve the efficacy of certain anti-cancer treatments. Among the most characterized probiotics, Lactobacillus rhamnosus GG (LGG) is currently utilized in clinics to alleviate diarrhea, mucositis or intestinal damage which might be associated with several triggers, including Clostridium difficile infections, inflammatory gut diseases, antibiotic consumption, chemotherapy or radiation therapy. Here, we investigate whether LGG cell-free supernatant (LGG-SN) might exert anti-proliferative activity toward colon cancer and metastatic melanoma cells. Moreover, we assess the potential adjuvant effect of LGG-SN in combination with anti-cancer drugs. METHODS: LGG-SN alone or in combination with either 5-Fuorouracil and Irinotecan was used to treat human colon and human melanoma cancer cell lines. Dimethylimidazol-diphenyl tetrazolium bromide assay was employed to detect cellular viability. Trypan blue staining, anti-cleaved caspase-3 and anti-total versus anti-cleaved PARP western blots, and annexin V/propidium iodide flow cytometry analyses were used to assess cell death. Flow cytometry measurement of cellular DNA content (with propidium iodide staining) together with qPCR analysis of cyclins expression were used to assess cell cycle. RESULTS: We demonstrate that LGG-SN is able to selectively reduce the viability of cancer cells in a concentration-dependent way. While LGG-SN does not exert any anti-proliferative activity on control fibroblasts. In cancer cells, the reduction in viability is not associated with apoptosis induction, but with a mitotic arrest in the G2/M phase of cell cycle. Additionally, LGG-SN sensitizes cancer cells to both 5-Fluorouracil and Irinotecan, thereby showing a positive synergistic action. CONCLUSION: Overall, our results suggest that LGG-SN may contain one or more bioactive molecules with anti-cancer activity which sensitize cancer cells to chemotherapeutic drugs. Thus, LGG could be proposed as an ideal candidate for ground-breaking integrated approaches to be employed in oncology, to reduce chemotherapy-related side effects and overcome resistance or relapse issues, thus ameliorating the therapeutic response in cancer patients.

Diagnostic value of neutrophil gelatinase-associated lipocalin/matrix metalloproteinase-9 pathway in transitional cell carcinoma of the bladder.

Neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP)-9, and NGAL/MMP-9 complex have been evaluated as diagnostic markers of several cancers, but results for bladder cancer are scanty. We evaluated these proteins in urine and serum of 89 patients with histologically confirmed bladder cancer and 119 cancer-free controls from a case-control study. Urinary concentrations were standardized on creatinine level. The performance of these proteins as cancer biomarkers was evaluated through the receiver operating characteristic (ROC) analysis. Urinary level of NGAL, MMP-9, and NGAL/MMP-9 complex was higher in current smokers, whereas no impact of dietary habits was observed. After adjusting for tobacco smoking, urinary concentration of MMP-9 was independently associated with cancer invasiveness, grading, and histological subtype, with elevated concentrations among T2-T4 and non-papillary bladder cancers. Conversely, NGAL and NGAL/MMP-9 complex were significantly higher in non-papillary than in papillary subtype. The pattern was less clear in serum, but correlation between urinary and serum concentration was poor, especially for Ta/is-T1 tumors. The ROC analysis confirmed that MMP-9 was the best marker (area under the ROC curve (AUC) = 0.68). Performances were much greater for muscle-invasive bladder cancers (AUC = 0.90), with elevated negative predictive values (97 %). The present study suggests that NGAL/MMP-9 pathway is associated with an aggressive phenotype of bladder cancer. The elevated negative predictive value of MMP-9 and NGAL/MMP-9 complex makes them candidate markers of exclusion test for bladder cancer. These proteins may be integrated in the surveillance of bladder cancer, thus diminishing patients' discomfort and improving compliance.

FBLN-3 as a biomarker of pleural plaques in workers occupationally exposed to carcinogenic fibers: a pilot study.

FBLN-3 has recently been proposed as a biomarker for malignant mesothelioma. A significantly increased standardized mortality rate from malignant mesothelioma has been reported in Biancavilla, Italy. Its cause has been identified in environmental exposure to fluoro-edenite. The aim of this study was to seek a correlation between plasma FBLN-3 concentration and pleural plaques in subjects exposed to fluoro-edenite and in a nonexposed control group. Pleural plaques was never detected in the control group, whereas it was found in 52% of exposed subjects. Median FBLN-3 concentrations were 12.96 and 5.29 ng/ml in the exposed and the control group, respectively (p < 0.001). FBLN-3 plasma levels exhibited a high predictive value for the presence of pleural plaques.

DNA Damage and Apoptosis as In-Vitro Effect Biomarkers of Titanium Dioxide Nanoparticles (TiO2-NPs) and the Food Additive E171 Toxicity in Colon Cancer Cells: HCT-116 and Caco-2.

This study investigated the DNA damage and apoptosis in colon cancer cells HCT-116 and Caco-2 induced by engineered titanium dioxide nanoparticles (TiO2-NPs) (60 nm) and titanium dioxide food additive E171. MTT assays showed that both chemical forms significantly reduced cancer cell viability in a dose-dependent manner. In particular the food additive E171 induced a pronounced inhibitory effect on the growth of HCT-116 and Caco-2 cell lines (E171 IC50: 3.45 mg/L for HTC-116 and 1.88 mg/L Caco-2; TiO2-NPs 60 nm IC50: 41.1 mg/L for HTC-116 and 14.3 mg/L for Caco-2). A low level of genotoxicity was observed in Caco-2 cells, especially when treated with TiO2 60 nm. Western blot analysis showed that HCT116 and Caco-2 treated cells did not overexpress apoptotic markers such as cleaved Caspase 3 and cleaved Parp. Moreover, further analysis by quantitative real-time PCR (qRT-PCR) showed that TiO2-NPs and E171 did not promote the expression of Bax or downregulation of Bcl-2, nor did they increase the Bax/Bcl-2 ratio. The assay data provide clear evidence that TiO2 can cause DNA damage but does not induce apoptosis or decrease long-term cell proliferation. In addition, the results show that E171 has a slightly higher level of cytotoxicity and genotoxicity. This suggests that exposure to E171 may be hazardous to health and that further research on biological effects is needed to promote safer practices in the use of this compound.

Overactivation of IL6 cis­signaling in leukocytes is an inflammatory hallmark of deep vein thrombosis.

Inflammation is a protective response of the body to various injuries, which is strictly regulated by a variety of factors, including immune cells and soluble mediators. However, dysfunction of this defensive mechanism often results in inflammation­driven diseases, such as deep vein thrombosis (DVT). The complex relationship between inflammatory cell activity and DVT has not been fully elucidated. The present study aimed to investigate the role of interleukin­6 (IL6) signaling transduction in DVT. To this aim, the expression levels of transmembrane isoforms of the IL6 receptor (IL6R) and the glycoprotein 130 responsible for the IL6 cis­signaling were evaluated in the peripheral blood mononuclear cells of patients with DVT and of healthy controls. The results indicated that leukocytes from patients with DVT exhibited overexpression of both IL6R and gp130 membrane isoforms and that these were strongly associated with the occurrence of DVT. Overall, the present findings indicated that IL6 cis­signaling may have a direct involvement in the leukocyte activation in DVT and may serve as a predictive biomarker of DVT development.

The PIK3CA H1047R Mutation Confers Resistance to BRAF and MEK Inhibitors in A375 Melanoma Cells through the Cross-Activation of MAPK and PI3K-Akt Pathways.

The targeting of the Mitogen-Activated Protein Kinase (MAPK) signalling pathway in melanoma improves the prognosis of patients harbouring the V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) mutation. However, a fraction of these patients may experience tumour progression due to resistance to targeted therapy. Mutations affecting the Phosphoinositol-3-Kinase (PI3K)-Akt pathway may favour the onset of drug resistance, suggesting the existence of a crosstalk between the MAPK and PI3K-Akt pathways. We hypothesized that the inhibition of both pathways may be a therapeutic option in resistant melanoma. However, conflicting data have been generated in this context. In this study, three different A375 cell melanoma models either overexpressing or not expressing the wild-type or mutated form of the PhosphatidylInositol-4,5-bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) gene were used to clarify the therapeutic response of melanoma to BRAF, Mitogen-Activated Protein Kinase Kinase 1 (MEK), and PI3K inhibitors in the presence of the PIK3CA H1047R mutation. Our data strongly support the notion that the crosstalk between the MAPK and PI3K-Akt pathways is one of the main mechanisms associated with melanoma development and progression and that the combination of MAPK and PI3K inhibitors may sensitize melanoma cells to therapy.

Balancing the Virulence and Antimicrobial Resistance in VISA DAP-R CA-MRSA Superbug.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) with intermediate resistance to Vancomycin (VISA) is reported worldwide. These strains frequently emerge among hospital-associated (HA)-MRSA and rarely within community-acquired (CA)-MRSA. Here, the genomic and transcriptomic adaptations distinguishing VISA daptomycin resistant (DAP-R) CA-MRSA, which emerged in a hospitalized patient under glycopeptide treatment, were explored. METHODS: Whole-genome sequencing, RNA-Seq and bioinformatics were carried out. RESULTS: Our CA-MRSA clustered in the USA400 lineage showing additional antimicrobial resistance (AMR) versus DAP and glycopeptides. Resistomics revealed adaptations related to glycopeptide, daptomycin and rifampin resistance (mprF nsSNPS and overexpression of glycopeptide and daptomycin-resistance related genes). Similar changes were detected in virulence traits (agrA HI-nsSNPs and toxin gene underexpression), in which a decrease was observed despite the abundance of virulence-related genes. Our results predicted a balance in adaptations, decreasing the virulence and biological costs to support the co-occurrence of extensive AMR in a hypervirulent genomic background. CONCLUSION: Our data show that VISA DAP-R CA-MRSA shifts the potential hypervirulent behavior of CA-MRSA towards the acquisition and maintenance of extensive AMR, by a decrease in virulence and biological costs mediated by a "compensatory modulatory mutation" silencing the Agr quorum-sensing cascade.

Co-Occurrence of Interleukin-6 Receptor Asp358Ala Variant and High Plasma Levels of IL-6: An Evidence of IL-6 Trans-Signaling Activation in Deep Vein Thrombosis (DVT) Patients.

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in several mechanisms, and the alteration of IL-6 signaling leads to the overactivation of various processes including immunity, inflammation, and hemostasis. Although IL-6 increase has been documented in venous thromboembolic diseases, the exact involvement of IL-6 signaling in deep vein thrombosis (DVT) has not been fully understood. Consequently, we investigated the involvement of IL-6 trans-signaling in inflammatory events occurring in DVT, focusing on the role of the interleukin-6 receptor (IL6-R) Asp358Ala variant. The circulating levels of IL-6, soluble IL6-R (sIL6-R), and soluble glycoprotein 130, as well as the Asp358Ala genotyping, were assessed in a consecutive cohort of DVT patients and healthy controls. The results indicated that IL-6 was higher in DVT compared to controls. Moreover, sIL6-R levels were strongly correlated to Asp358Ala variant in both groups, showing a high frequency of this mutation across all samples. Interestingly, our results showed a high frequency of both Asp358Ala mutation and raised IL-6 levels in DVT patients (OR = 21.32; p ≤ 0.01), highlighting that this mutation could explain the association between IL-6 overactivation and DVT outcome. Overall, this study represents a proof of concept for the targeting of IL-6 trans-signaling as a new strategy for the DVT adjuvant therapy.

Chemical Characterization and Quantification of Silver Nanoparticles (Ag-NPs) and Dissolved Ag in Seafood by Single Particle ICP-MS: Assessment of Dietary Exposure.

This study provides a first insight on the chemical characterization and quantification of silver nanoparticles (AgNPs) and dissolved Ag in processed canned seafood products, where food-grade edible silver (E174) is not intentionally added nor is the nanoparticle contained in the food contact material. The aim was to evaluate the bioaccumulation potential of AgNPs and to contribute to the assessment of AgNPs and ionic Ag human dietary intake from processed seafood. It is known how seafood, and in particular pelagic fish, is a precious nutritional source of unsaturated fatty acids, protein, and different micronutrients. Nevertheless, it may cause possible health problems due to the intake of toxic compounds coming from environmental pollution. Among emerging contaminants, AgNPs are widely applied in several fields such as biomedicine, pharmaceutical, food industry, health care, drug-gene delivery, environmental study, water treatments, and many others, although its primary application is in accordance with its antimicrobial property. As a consequence, AgNPs are discharged into the aquatic environment, where the colloidal stability of these NPs is altered by chemical and physical environmental parameters. Its toxicity was demonstrated in in-vitro and in-vivo studies, although some findings are controversial because toxicity depends by several factors such as size, concentration, chemical composition, surface charge, Ag+ ions released, and hydrophobicity. The new emerging technique called single-particle inductively coupled plasma mass spectrometry (spICP-MS) was applied, which allows the determination of nanoparticle number-based concentration and size distribution, as well as the dissolved element. Our findings highlighted comparable mean sizes across all species analysed, although AgNPs concentrations partly follow a trophic level-dependent trend. The low mean size detected could be of human health concern, since, smaller is the diameter higher is the toxicity. Dietary intake from a meal calculated for adults and children seems to be very low. Although seafood consumption represents only a small part of the human total diet, our findings represent a first important step to understand the AgNPs dietary exposure of the human population. Further studies are needed to characterize and quantify AgNPs in a large number of food items, both processing and not, and where AgNPs are added at the industrial level. They will provide a realistic exposure assessment, useful to understand if AgNPs toxicity levels observed in literature are close to those estimable through food consumption and implement data useful for risk assessors in developing AgNPs provisional tolerable daily intake.

Identification of Novel MicroRNAs and Their Diagnostic and Prognostic Significance in Oral Cancer.

Background: Oral cancer is one of the most prevalent cancers worldwide. Despite that the oral cavity is easily accessible for clinical examinations, oral cancers are often not promptly diagnosed. Furthermore, to date no effective biomarkers are available for oral cancer. Therefore, there is an urgent need to identify novel biomarkers able to improve both diagnostic and prognostic strategies. In this context, the development of innovative high-throughput technologies for molecular and epigenetics analyses has generated a huge amount of data that may be used for the identification of new cancer biomarkers. Methods: In the present study, GEO DataSets and TCGA miRNA profiling datasets were analyzed in order to identify miRNAs with diagnostic and prognostic significance. Furthermore, several computational approaches were adopted to establish the functional roles of these miRNAs. Results: The analysis of datasets allowed for the identification of 11 miRNAs with a potential diagnostic role for oral cancer. Additionally, eight miRNAs associated with patients' prognosis were also identified; six miRNAs predictive of patients' overall survival (OS) and one, hsa-miR-let.7i-3p, associated with tumor recurrence. Conclusion: The integrated analysis of different miRNA expression datasets allows for the identification of a set of miRNAs that, after validation, may be used for the early detection of oral cancers.

Prognostic significance of deregulated microRNAs in uveal melanomas.

Uveal melanoma (UM) represents the most frequent primary tumor of the eye. Despite the development of new drugs and screening programs, the prognosis of patients with UM remains poor and no effective prognostic biomarkers are yet able to identify high­risk patients. Therefore, in the present study, microRNA (miRNA or miR) expression data, contained in the TCGA UM (UVM) database, were analyzed in order to identify a set of miRNAs with prognostic significance to be used as biomarkers in clinical practice. Patients were stratified into 2 groups, including tumor stage (high­grade vs. low­grade) and status (deceased vs. alive); differential analyses of miRNA expression among these groups were performed. A total of 20 deregulated miRNAs for each group were identified. In total 7 miRNAs were common between the groups. The majority of common miRNAs belonged to the miR­506­514 cluster, known to be involved in UM development. The prognostic value of the 20 selected miRNAs related to tumor stage was assessed. The deregulation of 12 miRNAs (6 upregulated and 6 downregulated) was associated with a worse prognosis of patients with UM. Subsequently, miRCancerdb and microRNA Data Integration Portal bioinformatics tools were used to identify a set of genes associated with the 20 miRNAs and to establish their interaction levels. By this approach, 53 different negatively and positively associated genes were identified. Finally, DIANA­mirPath prediction pathway and Gene Ontology enrichment analyses were performed on the lists of genes previously generated to establish their functional involvement in biological processes and molecular pathways. All the miRNAs and genes were involved in molecular pathways usually altered in cancer, including the mitogen­activated protein kinase (MAPK) pathway. Overall, the findings of the presents study demonstrated that the miRNAs of the miR­506­514 cluster, hsa­miR­592 and hsa­miR­199a­5p were the most deregulated miRNAs in patients with high­grade disease compared to those with low­grade disease and were strictly related to the overall survival (OS) of the patients. However, further in vitro and translational approaches are required to validate these preliminary findings.

Gut Microbiota and Cancer: From Pathogenesis to Therapy.

Cancer is a multifactorial pathology and it represents the second leading cause of death worldwide. In the recent years, numerous studies highlighted the dual role of the gut microbiota in preserving host's health. Gut resident bacteria are able to produce a number of metabolites and bioproducts necessary to protect host's and gut's homeostasis. Conversely, several microbiota subpopulations may expand during pathological dysbiosis and therefore produce high levels of toxins capable, in turn, to trigger both inflammation and tumorigenesis. Importantly, gut microbiota can interact with the host either modulating directly the gut epithelium or the immune system. Numerous gut populating bacteria, called probiotics, have been identified as protective against the genesis of tumors. Given their capability of preserving gut homeostasis, probiotics are currently tested to help to fight dysbiosis in cancer patients subjected to chemotherapy and radiotherapy. Most recently, three independent studies show that specific gut resident species may potentiate the positive outcome of anti-cancer immunotherapy. The highly significant studies, uncovering the tight association between gut microbiota and tumorigenesis, as well as gut microbiota and anti-cancer therapy, are here described. The role of the Lactobacillus rhamnosus GG (LGG), as the most studied probiotic model in cancer, is also reported. Overall, according to the findings here summarized, novel strategies integrating probiotics, such as LGG, with conventional anti-cancer therapies are strongly encouraged.

MMP-9 as a Candidate Marker of Response to BRAF Inhibitors in Melanoma Patients With BRAFV600E Mutation Detected in Circulating-Free DNA.

The BRAFV600E mutation is associated with melanoma development and its detection in circulating-free DNA cannot be observed in all melanoma patients harboring this mutation in tumor specimens. Beside the circulating-free DNA BRAFV600E mutation, other markers of therapeutic response should be identified. Matrix metalloproteinase-9 (MMP-9) could be one of them as its role as indicator of invasiveness in melanoma have been explored. In this study, MMP-9 was evaluated in melanoma cells after treatment with dabrafenib. In vitro data were validated in 26 melanoma patients, of which 14 treated with BRAF inhibitor alone and 12 treated with both BRAF and MEK inhibitors, by ELISA assay and droplet digital PCR for measuring MMP-9 serum levels and circulating-free DNA BRAFV600E mutation, respectively. Statistical analyses were performed to evaluate the prognostic significance of MMP-9, progression-free survival (PFS) and overall survival (OS) according to the BRAFV600E mutation and MMP-9 levels. The performed analyses showed that MMP-9 and pEKR1-2 were statistically down-regulated in melanoma cells after treatment with dabrafenib. Circulating-free DNA BRAFV600E mutation was detected in 11 out of 26 melanoma patients showing higher levels of MMP-9 compared to those with undetectable BRAFV600E mutation. Furthermore, higher levels of MMP-9 and circulating-free DNA BRAFV600E mutation were associated with lower PFS and OS. Finally, the monitoring of therapy showed that MMP-9 significantly decreased at T1 and T2, but not at T-last, for the patients with detectable circulating-free DNA BRAFV600E mutation. In conclusion, high levels of MMP-9 and circulating-free DNA BRAFV600E mutation are associated with poor PFS and OS. MMP-9 may represent a promising indicator of response to BRAF inhibitors in combination with the detection of BRAFV600E mutation.

Cutaneous melanoma: From pathogenesis to therapy (Review).

In less than 10 years, melanoma treatment has been revolutionized with the approval of tyrosine kinase inhibitors and immune checkpoint inhibitors, which have been shown to have a significant impact on the prognosis of patients with melanoma. The early steps of this transformation have taken place in research laboratories. The mitogen­activated protein kinase (MAPK) pathway, phosphoinositol­3­kinase (PI3K) pathway promote the development of melanoma through numerous genomic alterations on different components of these pathways. Moreover, melanoma cells deeply interact with the tumor microenvironment and the immune system. This knowledge has led to the identification of novel therapeutic targets and treatment strategies. In this review, the epidemiological features of cutaneous melanoma along with the biological mechanisms involved in its development and progression are summarized. The current state­of­the­art of advanced stage melanoma treatment strategies and the currently available evidence of the use of predictive and prognostic biomarkers are also discussed.

Epigenetic alterations and occupational exposure to benzene, fibers, and heavy metals associated with tumor development (Review).

The chronic occupational exposure to contaminants and carcinogens leads to the development of cancer. Over the past decades, many carcinogens have been found in the occupational environment and their presence is often associated with an increased incidence of cancer. According to the International Agency for Research on Cancer (IARC), the majority of carcinogens are classified as 'probable' and 'possible' human carcinogens, while, direct evidence of carcinogenicity is provided in epidemiological and experimental studies. Additionally, accumulating evidence suggests that epigenetic alterations may be early indicators of genotoxic and non-genotoxic carcinogen exposure. In the present review, the relationship between exposures to benzene, mineral fibers, metals and epigenetic alterations are discussed as the most important cancer risk factors during work activities.

Lactobacillus rhamnosus GG: An Overview to Explore the Rationale of Its Use in Cancer.

Cancer is the second leading cause of death in the western world. In the era of precision medicine, a significant number of cancer patients can be cured with several anti-cancer therapeutic regimens. However, therapy failure may be caused by treatment side effects, such as diarrhea, especially occurring in patients with gastrointestinal or pelvic malignancies. In particular, diarrhea is one of the most frequent gastrointestinal toxicity during cancer treatment and it can result from nearly bot chemo- and radio-therapeutic strategies currently used. Diarrhea has a serious impact on patients' quality of life and treatment dosing and schedule modification due to its severity can negatively influence treatment outcomes. In this context, probiotics may play an interesting role in several human diseases with an inflammatory bowel involvement and, among these, Lactobacillus rhamnosus GG (LGG) is one of the most characterized and utilized. In particular, LGG is able to reverse intestinal dysbiosis and moderate diarrhea. Moreover, preclinical studies have documented its effects in reducing chronic inflammation associated with cancer development. This review summarizes the preclinical results of LGG on cancer cells proliferation and tumor invasion as well as the potential role of LGG use in cancer patients for the prevention and management of diarrhea associated with cancer treatment. Overall, these encouraging data support further investigation on the use of LGG in stratified patients undergoing specific therapeutic protocols, including chemotherapy and pelvic radiotherapy, in order to reduce the development of severe diarrhea and thus improve the adherence to the therapy and patients' quality of life.

Low levels of inflammation and the absence of subclinical atherosclerosis in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Patients with RA have an increased risk for the development of cardiovascular diseases, however, the pathophysiological mechanisms of arterial complications in RA remain to be fully elucidated. Understanding the early markers of vascular damage may aid in preventing cardiovascular complications in patients with RA. The current study investigated this by recruiting 30 patients with RA and 30 healthy subjects. Intima­media thickness (IMT) was used to detect the presence of atherosclerotic disease and was measured in the carotid and femoral arteries. Tumor necrosis factor α, interleukin­6 (IL­6), IL­8, IL­10 and matrix metalloproteinase­2 were measured as markers of inflammation. An IMT ≥0.9 mm was observed in 7/30 patients with RA, however, no significant differences between patients with RA and the controls were observed in the inflammatory markers analyzed. Of note, these results indicated that the appropriate management of RA may have affected the inflammatory status of these patients and consequently may have impacted upon subclinical atherosclerosis.

MMP-9 overexpression is associated with intragenic hypermethylation of MMP9 gene in melanoma.

Tumor spreading is associated with the degradation of extracellular matrix proteins, mediated by the overexpression of matrix metalloproteinase 9 (MMP-9). Although, such overexpression was linked to epigenetic promoter methylation, the role of intragenic methylation was not clarified yet. Melanoma was used as tumor model to investigate the relationship between the DNA intragenic methylation ofMMP9 gene and MMP-9 overexpression at transcriptional and protein levels. Computational analysis revealed DNA hypermethylation within the intragenic CpG-2 region of MMP9 gene in melanoma samples with high MMP-9 transcript levels. In vitro validation showed that CpG-2 hotspot region was hypermethylated in the A375 melanoma cell line with highest mRNA and protein levels of MMP-9, while low methylation levels were observed in the MEWO cell line where MMP-9 was undetectable. Concordant results were demonstrated in both A2058 and M14 cell lines. This correlation may give further insights on the role of MMP-9 upregulation in melanoma.

Fluoro-edenite induces fibulin-3 overexpression in non-malignant human mesothelial cells.

Exposure to asbestos is associated with the development of mesothelioma. In addition to asbestos, other fibers have been identified as risk factors for malignant and non-malignant diseases of the lungs. Among these, fluoro-edenite (FE) was found in patients from Biancavilla (Sicily, Italy) with pleural and lung disease, suggesting its role for tumor expansion. In this context, the identification of early biomarkers useful for the diagnosis of cancer is mandatory. Fibulin-3 represents an important marker for the diagnosis of mesothelioma. However, it remains to be determined whether it is directly associated with exposure to asbestos-like fibers. In the present study, peripheral blood levels of fibulin-3 from 40 asbestos-exposed workers were compared with those detected in 27 street cleaners from Biancavilla. Intriguingly, the results showed that fibulin-3 levels were higher in the group of street cleaners compared with those of the asbestos-exposed workers, suggesting that these workers used the personal protective equipment according to the current regulations. These data suggest that subjects exposed to FE should be monitored for the risk of mesothelioma. FE and volcanic particulates are probably contained within dust inhaled by street cleaners from Biancavilla during their work activities. Based on these criteria, in this study, such fibers were used to treat mesothelial cells (MeT5A) in order to verify whether fibulin-3 levels are affected by these treatments. The results showed that only treatment with FE was associated with fibulin-3 overexpression at both the transcript and protein levels. It was previously demonstrated that mesothelial cells exhibited low levels of p27 following treatment with FE. Notably, p27 downregulation is associated with stathmin upregulation in cancer, conferring an aggressive phenotype of tumor cells. This observation prompted us to perform a computational evaluation demonstrating the activation of stathmin in lung cancer in patients exposed to asbestos. Overall, it can be speculated that both fibulin-3 and stathmin overexpression may be associated with the malignant transformation of mesothelial cells following exposure to asbestos-like fibers.

Computational identification of microRNAs associated to both epithelial to mesenchymal transition and NGAL/MMP-9 pathways in bladder cancer.

Bladder cancer is one of the leading cancer of the urinary tract. It is often diagnosed at advanced stage of the disease. To date, no specific and effective early detection biomarkers are available. Cancer development and progression are associated with the involvement of both epithelial-mesenchymal transition (EMT) and tumor microenvironment of which NGAL/MMP-9 complex represents the main player in bladder cancer. It is known that change in microRNAs (miRNAs) expression may result in gene modulation. Therefore, the identification of specific miRNAs associated with EMT pathway and NGAL/MMP-9 complex may be useful to detect the development of bladder cancer at early stages.On this ground, the expression levels of miRNAs in public available datasets of bladder cancer containing data of non-coding RNA profiling was evaluated. This analysis revealed a group of 16 miRNAs differentially expressed between bladder cancer patients and related healthy controls. By miRNA prediction tool (mirDIP), the relationship between the identified miRNAs and the EMT genes was established. Using the DIANA-mirPath (v.2) software, miRNAs, able to modulate the expression of NGAL and MMP-9 genes, were recognized.The results of this study provide evidence that the downregulated hsa-miR-145-5p and hsa-miR-214-3p may modulate the expression of both EMT and NGAL/MMP-9 pathways. Therefore, further validation analyses may confirm the usefulness of these selected miRNAs for predicting the development of bladder cancer at the early stage of the disease.