Iacopo Sardi

Expression of matrix metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 9 in carcinoma of the head and neck

Numerous reports have documented a direct involvement of matrix metalloproteinase (MMP) overexpression in the development and progression of head and neck squamous cell carcinoma (HNSCC). In this study, the authors examined whether the expression of MMPs in HNSCC is correlated with other steps involved in tumor growth and metastasis, like angiogenesis, activation the nitric oxide (NO) pathway, and alteration of the p53 tumor suppressor gene.

Cyclooxygenase-2 Activation Mediates the Proangiogenic Effect of Nitric Oxide in Colorectal Cancer

Purpose: Up-regulation of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes has been reported in colorectal cancer. We aimed at evaluating the possible interaction between the nitric oxide and COX-2 pathways, and its effect on promoting tumor angiogenesis. Experimental Design: Expression of iNOS, COX-2, vascular endothelial growth factor (VEGF), and CD31 was analyzed in tumor samples and corresponding normal mucosa obtained from 46 surgical specimens. We also evaluated iNOS activity, prostaglandin E2 (PGE2), cyclic GMP and cyclic AMP production in the same specimens. Nitrite/nitrate levels, and PGE2 and VEGF production were assessed in HCT116 and HT29 colon cancer cell lines after induction and selective inhibition of the two enzyme pathways. Results: A significant correlation was found between iNOS and COX-2 immunohistochemical expression. PGE2 production significantly correlated with iNOS activity and cGMP levels. A significant correlation was also found among PGE2 production, microvessel density, and VEGF expression. Coinduction of both iNOS and COX-2 activities occurred after lipopolysaccharide (LPS) and epidermal growth factor (EGF) treatment in HCT116 and HT29 cells. Inhibition of iNOS by 1400W significantly reduced both LPS- and EGF-induced PGE2 production. Treatment with LPS, EGF, and arachidonic acid significantly increased VEGF production in the iNOS-negative/COX-2-positive HT29 cells. This effect was completely reversed by treatment with the selective COX-2 inhibitor celecoxib. Conclusions: Our data showed a prominent role of nitric oxide in stimulating COX-2 activity in colorectal cancer. This interaction is likely to produce a cooperative effect in promoting angiogenesis through PGE2-mediated increase in VEGF production.

Inducible Nitric Oxide Synthase Expression in Human Colorectal Cancer : Correlation with Tumor Angiogenesis

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.

Exposure to global system for mobile communication (GSM) cellular phone radiofrequency alters gene expression, proliferation, and morphology of human skin fibroblasts.

Human skin fibroblasts were exposed to global system for mobile communication (GSM) cellular phone radiofrequency for 1 h. GSM exposure induced alterations in cell morphology and increased the expression of mitogenic signal transduction genes (e.g., MAP kinase kinase 3, G2/mitotic-specific cyclin G1), cell growth inhibitors (e.g., transforming growth factor-beta), and genes controlling apoptosis (e.g., bax). A significant increase in DNA synthesis and intracellular mitogenic second messenger formation matched the high expression of MAP kinase family genes. These findings show that these electromagnetic fields have significant biological effects on human skin fibroblasts.

Hyperfractionated Accelerated Radiotherapy in the Milan Strategy for Metastatic Medulloblastoma

PURPOSE With a view to improving the prognosis for patients with metastatic medulloblastoma, we tested the efficacy and toxicity of a hyperfractionated accelerated radiotherapy (HART) regimen delivered after intensive sequential chemotherapy. PATIENTS AND METHODS Between 1998 and 2007, 33 consecutive patients received postoperative methotrexate (8 g/m(2)), etoposide (2.4 g/m(2)), cyclophosphamide (4 g/m(2)), and carboplatin (0.8 g/m(2)) in a 2-month schedule, then HART with a maximal dose to the neuraxis of 39 Gy (1.3 Gy/fraction, 2 fractions/d) and a posterior fossa boost up to 60 Gy (1.5 Gy/fraction,2 fractions/d). Patients with persistent disseminated disease before HART were consolidated with two myeloablative courses and circulating progenitor cell rescue. RESULTS Patients were classified as having M1 (n = 9), M2 (n = 6), M3 (n = 17), and M4 (n = 1) disease. Seven patients younger than 10 years old who achieved complete response after chemotherapy received a lower dose to the neuraxis (31.2 Gy). Twenty-two of the 32 assessable patients responded to chemotherapy; disease was stable in five patients and progressed in five patients. One septic death occurred before radiotherapy. Eight patients experienced relapse after a median of 12 months. Fourteen of the 33 patients underwent consolidation therapy after HART. With a median 82-month survivor follow-up, the 5-year event-free, progression-free, and overall survival rates were 70%, 72%, and 73%, respectively. No severe clinical complications of HART have emerged so far. CONCLUSION HART after intensive postoperative chemotherapy, followed by myeloablative chemotherapy in selected cases, proved feasible in children with metastatic medulloblastoma. The results of our treatment compare favorably with other series treated using conventional therapies.

Down-regulation of nitric oxide synthase-2 and cyclooxygenase-2 pathways by p53 in squamous cell carcinoma

The goal of this study was to analyze the correlation between inducible nitric oxide synthase (iNOS) and COX-2 activities and p53 gene status in head and neck squamous cell carcinomas (HNSCCs) in vivo and in vitro. In a series of 43 HNSCCs we observed an up-regulation of both iNOS and COX-2 pathways in tumor tissues and both activities were correlated each other (rs = 0.612 and P = 0.0002). We also found that p53-mutated HNSCCs (25 cases, 58.1%) showed higher levels of iNOS activity and cGMP in comparison with wild-type p53 tumors (18 cases, 41.9%) (P = 0.0005 and P = 0.01), as well as higher iNOS immunohistochemical expression (P = 0.03). Analogously, higher PgE2 levels were documented in p53-mutated HNSCCs when compared with wild-type p53 tumors (P = 0.015) and COX-2 protein expression was higher in p53-mutated HNSCCs (P = 0.007). A431 cancer cells expressing a p53 temperature-sensitive mutant showed an approximately 1.9- and 2.6-fold decrease in spontaneous NO(2-)/NO(3-) and PgE2 synthesis at permissive temperature, respectively, when compared with the same cells at nonpermissive temperature (P <or= 0.001). Basal levels of iNOS and COX-2 proteins and mRNAs were markedly suppressed by restoration of p53 activity. Our results indicate that p53 gene mutation(s) may be responsible for iNOS and COX-2 up-regulation frequently observed in HNSCCs and suggest that restoration of wild-type p53 expression may interfere with tumor growth by inhibiting iNOS and COX-2 pathways.

Inducible nitric oxide synthase expression in benign and malignant cutaneous melanocytic lesions.

Nitric oxide (NO) is synthesized by nitric oxide synthases (NOS) and plays an important role in tumour growth. In this study, inducible NOS (iNOS) expression was evaluated by immunohistochemistry in 34 melanocytic naevi (13 common melanocytic naevi, six Spitz naevi, and 15 so‐called ‘dysplastic naevi’), ten cutaneous melanomas in situ, 50 stage I invasive melanomas, and eight subcutaneous metastases of melanoma. In addition, four samples of melanocytic naevi and four samples of invasive melanomas were collected in order to perform western blot and northern blot analysis. By immunohistochemistry, melanocytic naevi never expressed iNOS. Among cases of melanoma in situ, two were negative, seven displayed staining in less than 20% of melanoma cells, and positivity was observed in 21–50% of melanoma cells in only one case. iNOS expression was detected in 46 out of 50 invasive melanomas (92%). Among these cases, 18 showed positivity in less than 20% of melanoma cells, 18 showed positivity in 21–50% of melanoma cells, and ten showed iNOS expression in more than 50% of cells. Statistical analysis revealed a significant difference in iNOS expression between melanocytic naevi and cutaneous melanomas (p<0.001). In addition, iNOS expression was significantly higher in invasive melanomas than in melanomas in situ (p=0.01). Among primary cutaneous melanomas, no significant correlation was found between iNOS expression and histopathological parameters (histotype, level, thickness and presence of regression/inflammatory infiltrate) and disease‐specific survival. In subcutaneous melanoma metastases, iNOS expression was diffuse in more than 50% of cells. Statistical analysis revealed that subcutaneous melanoma metastases showed greater iNOS immunoreactivity than invasive melanomas (p=0.02). Molecular analyses confirmed that iNOS mRNA and protein were highly expressed in melanoma samples. In conclusion, iNOS was constantly absent in melanocytic naevi, whereas it was frequently expressed in melanomas, with up‐regulation of the enzyme paralleling tumour progression. These data suggest that iNOS may play a role in the malignant transformation of melanocytes and in tumour growth. In addition, iNOS may be useful as an immunohistochemical marker for malignant melanocytic lesions. Copyright

EFFECT OF 0.2 T STATIC MAGNETIC FIELD ON HUMAN NEURONS : REMODELING AND INHIBITION OF SIGNAL TRANSDUCTION WITHOUT GENOME INSTABILITY

We describe the effect of the static magnetic field generated by a 0.2 T magnetic resonance tomograph on a normal human neuronal cell culture (FNC-B4). After 15 min exposure cells showed dramatic changes of morphology: they formed vortexes of cells and exposed branched neurites featuring synaptic buttons. At the same time, thymidine incorporation and inositol lipid signaling were significantly reduced. Control (sham exposed) or non-neuronal cells (mouse leukemia, and human breast carcinoma cells) did not show any alteration following exposure. Endothelin-1 release from FNC-B4 cells was also dramatically reduced after 5 min exposure. However, PCR analysis of 12 DNA microsatellites selected as gauges of genome instability, did not reveal any alteration following exposure, thus ruling out a direct effect of the magnetic field on DNA stability. These data can be interpreted as a specific effect of the static magnetic field on human neuronal cells and are consistent with the induction of remodeling and differentiation; they demonstrate that fields below 0.5 T have significant biological effects on human neurons.

Plasmacytoid urothelial carcinoma of the urinary bladder: clinicopathologic, immunohistochemical, ultrastructural, and molecular analysis of a case series.

A plasmacytoid variant of urothelial carcinoma has been recently recognized in the World Health Organization classification system. This is characterized by a discohesive growth of plasmacytoid cells with eccentric nuclei, extending in the bladder wall and often in the perivesical adipose tissue. Herein, we report the clinicopathologic, immunohistochemical, ultrastructural, and molecular features of a series of plasmacytoid urothelial carcinoma of the urinary bladder. Four bladder carcinomas characterized by epithelial cells with morphologic appearance resembling plasma cells were evaluated at the immunohistochemical, electron microscopic, and molecular genetic levels. Tumor cells stained with cytokeratins, epithelial membrane antigen, GATA-3 (endothelial transcription factor 3), CD15, p53, and p16. In addition, malignant cells strongly stained with CD138 in all the cases, whereas leukocyte common antigen and multiple myeloma 1/interferon regulatory factor 4 were completely negative, nor immunoreactivity was seen for either κ or λ light chains. The electron microscopic examination showed the presence of divergent squamous and glandular differentiation. At variance with conventional urothelial carcinoma, the analysis of exons 4-9 of TP53 gene revealed no alteration in all the 4 tumors tested, and this can be of value in choosing additional chemotherapy after surgery. Plasmacytoid carcinoma of the bladder is a tumor entity, which can be characterized by specific immunohistochemical markers, including positivity for GATA-3, and presents phenotypic and genotypic peculiarities.

Correlation between nitric oxide and cyclooxygenase-2 pathways in head and neck squamous cell carcinomas.

We investigated the interactions between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) pathways in head and neck squamous cell carcinomas (HNSCCs) and in two carcinoma cell lines. HNSCCs showed an up-regulation of both pathways which were strongly correlated with each other (p=0.02) and with tumor vascularization (p=0.0001 and p=0.008, respectively). In carcinoma cells, Escherichia coli lipopolysaccharide (LPS) and EGF treatment up-regulated both pathways. NOS inhibitor N(G)-monomethyl-L-arginine methyl ester (L-NAME) inhibited this up-regulation. LPS or EGF induced iNOS expression that was not altered by NOS or COX-2 inhibitors. Conversely, LPS or EGF promoted COX-2 expression that was decreased by L-NAME. The NO donor S-nitroso-acetyl-penicillamine (SNAP) up-regulated COX-2 pathway and this effect was reduced by the guanylate cyclase inhibitor methylene blue. Thus, in squamous carcinoma cells, NO increases the activity of COX-2 pathway and this effect is probably mediated by endocellular cGMP level, with potential implications on tumor growth, angiogenesis, and therapy.