Romina Rocchetti

Cloning and characterization of a senescence inducing and class II tumor suppressor gene in ovarian carcinoma at chromosome region 6q27.

Cytogenetic, molecular and functional analysis has shown that chromosome region 6q27 harbors a senescence inducing gene and a tumor suppressor gene involved in several solid and hematologic malignancies. We have cloned at 6q27 and characterized the RNASE6PL gene which belongs to a family of cytoplasmic RNases highly conserved from plants, to man. Analysis of 55 primary ovarian tumors and several ovarian tumor cell lines indicated that the RNASE6PL gene is not mutated in tumor tissues, but its expression is significantly reduced in 30% of primary ovarian tumors and in 75% of ovarian tumor cell lines. The promoter region of the gene was unaffected in tumors cell lines. Transfection of RNASE6PL cDNA into HEY4 and SG10G ovarian tumor cell lines suppressed tumorigenicity in nude mice. When tumors were induced by RNASE6PL-transfected cells, they completely lacked expression of RNASE6PL cDNA. Tumorigenicity was suppressed also in RNASE6PL-transfected pRPcT1/H6cl2T cells, derived from a human/mouse monochromosomic hybrid carrying a human chromosome 6 deleted at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% of the clones of XP12ROSV, a Xeroderma pigmentosum SV40-immortalized cell line, transfected with RNASE6PL cDNA, developed a marked senescence process during in vitro growth. We therefore propose that RNASE6PL may be a candidate for the 6q27 senescence inducing and class II tumor suppressor gene in ovarian cancer.

Prognostic value analysis of urokinase-type plasminogen activator receptor in oral squamous cell carcinoma: an immunohistochemical study.

BackgroundOral squamous cell carcinoma (OSCC) represents the most common oral malignancy. Despite recent advances in therapy, up to 50% of the cases have relapse and/or metastasis. There is therefore a strong need for the identification of new biological markers able to predict the clinical behaviour of these lesions in order to improve quality of life and overall survival. Among tumour progression biomarkers, already known for their involvement in other neoplasia, a crucial role is ascribed to the urokinase-type plasminogen activator receptor (uPAR), which plays a multiple role in extracellular proteolysis, cell migration and tissue remodelling not only as a receptor for the zymogen pro-uPA but also as a component for cell adhesion and as a chemoattractant. The purpose of this study was to gain information on the expression of uPAR in OSCC and to verify whether this molecule can have a role as a prognostic/predictive marker for this neoplasia.MethodsIn a retrospective study, a cohort of 189 OSCC patients was investigated for uPAR expression and its cellular localization by immunohistochemistry. As standard controls, 8 normal oral mucosal tissues free of malignancy, obtained from patients with no evidence or history of oral cavity tumours, were similarly investigated. After grouping for uPAR expression, OSCCs were statistically analyzed for the variables age, gender, histological grading (G), tumour size, recurrence, TNM staging and overall survival rate.ResultsIn our immunohistochemical study, 74 cases (39.1%) of OSCC showed a mostly cytoplasmic positivity for uPAR, whereas 115 were negative. uPAR expression correlated with tumour differentiation grade and prognosis: percentage of positive cases was the greatest in G3 (70.4%) and patients positives for uPAR expression had an expectation of life lower than those for uPAR negatives.ConclusionThe results obtained in this study suggest a role of uPAR as a potential biomarker useful to identify higher risk subgroups of OSCC patients.

Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

Background/Aims: Head and neck squamous cell carcinoma (HNSCC) ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs), has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.

The role of peroxisome proliferator-activated receptor γ in bladder cancer in relation to angiogenesis and progression

Peroxisome proliferator-activated receptor gamma (PPAR gamma) immunohistochemical expression was analyzed in 75 human bladder tumor specimens, where the expression of some angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PDECGF), and tumor progression markers, such as epidermal growth factor receptor (EGFr), p16, mutated p53, and normal pRB, were also analyzed. The results were then compared to the clinical and pathological characteristics of the disease. PPAR gamma was expressed more significantly in papillary tumors than in solid cancers, and its presence was associated with statistical significance to low incidence of tumor recurrence or progression. This significant association was observed also when PPAR gamma was expressed in the presence of PDECGF, which resulted, when considered alone, to an angiogenic factor typical of solid cancers and appeared related to poor prognosis. In the presence of bFGF, on the contrary, PPAR gamma expression no longer resulted to a significant association with low incidence of tumor recurrence or progression, suggesting a possible worsening role of this angiogenic factor, typical of papillary cancers, in its interaction with PPAR gamma.

RNA-Mediated Gene Silencing of Nicotinamide N-Methyltransferase Is Associated with Decreased Tumorigenicity in Human Oral Carcinoma Cells

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite progress in the treatment of OSCC, overall survival has not improved substantially in the last three decades. Therefore, identification of reliable biomarkers becomes essential to develop effective anti-cancer therapy. In this study, we focused on the enzyme Nicotinamide N-methyltransferase (NNMT), which plays a fundamental role in the biotransformation of many xenobiotics. Although several tumors have been associated with abnormal NNMT expression, its role in cancer cell metabolism remains largely unknown. In this report, 7 human oral cancer cell lines were examined for NNMT expression by Real-Time PCR, Western blot and HPLC-based catalytic assay. Subsequently, we evaluated the in vitro effect of shRNA-mediated silencing of NNMT on cell proliferation. In vivo tumorigenicity of oral cancer cells with stable knockdown of NNMT was assayed by using xenograft models. High expression levels of NNMT were found in PE/CA PJ-15 cells, in keeping with the results of Western blot and catalytic activity assay. PE/CA PJ-15 cell line was stably transfected with shRNA plasmids against NNMT and analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and soft agar Assays. Transfected and control cells were injected into athymic mice in order to evaluate the effect of NNMT silencing on tumor growth. NNMT downregulation resulted in decreased cell proliferation and colony formation ability on soft agar. In athymic mice, NNMT silencing induced a marked reduction in tumour volume. Our results show that the downregulation of NNMT expression in human oral carcinoma cells significantly inhibits cell growth in vitro and tumorigenicity in vivo. All these experimental data seem to suggest that NNMT plays a critical role in the proliferation and tumorigenic capacity of oral cancer cells, and its inhibition could represent a potential molecular approach to the treatment of oral carcinoma.

Inhibiting proliferation in KB cancer cells by RNA interference-mediated knockdown of nicotinamide N-methyltransferase expression.

The enzyme Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide and other pyridines, playing a pivotal role in the biotransformation and detoxification of many drugs and xenobiotic compounds. Several tumours have been associated with abnormal NNMT expression, however its role in tumour development remains largely unknown. In this study we investigated expression levels of Nicotinamide N-methyltransferase in a cancer cell line and we evaluated the effect of shRNA-mediated silencing of NNMT on cell proliferation. Cancer cells were examined for NNMT expression by semiquantitative RT-PCR and Western blot analysis. A HPLC-based catalytic assay was performed to assess enzyme activity. Cells were transfected with four shRNA plasmids against NNMT and control cells were treated with transfection reagent only (mock). The efficiency of gene silencing was detected by Real-Time PCR and Western blot analysis. MTT cell proliferation assay and the soft agar colony formation assay were then applied to investigate the functional changes in cancerous cell. NNMT mRNA was detected in cancer cells, showing a very high expression level. In keeping with the results of RT-PCR analysis, the protein level and NNMT enzyme activity were particularly high in KB cells. ShRNA vectors targeted against NNMT efficiently suppressed gene expression, showing inhibition observed at both the mRNA and protein levels. Down-regulation of NNMT significantly inhibited cell proliferation and decreased colony formation ability on soft agar. The present data support the hypothesis that the enzyme plays a role in tumour expansion and its inhibition could represent a possible molecular approach to the treatment of cancer.

Analysis of tissue and salivary nicotinamide N-methyltransferase in oral squamous cell carcinoma: basis for the development of a noninvasive diagnostic test for early-stage disease.

Abstract Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer worldwide. In recent decades, the 5-year mortality rate is approximately 50% around the world. As reliable biomarkers of oral cancer are still lacking, it is necessary to identify new target molecules for early diagnosis, effective therapy, and monitoring of the disease. In the present work, we focused on the expression of the enzyme nicotinamide N-methyltransferase (NNMT). We analyzed enzyme activity in 37 paired tumor and non-tumor tissues and found that activity levels are significantly higher in tumor compared with adjacent normal oral mucosa. Interestingly, oral epithelium surrounding tumor of unfavorable cases (N+) seems to display higher activity levels compared with that of favorable ones (N0). Western blot analyses were performed to evaluate protein levels in saliva samples from patients with OSCC and healthy subjects. Preliminary results indicated an up-regulation of salivary NNMT in tumor. This study shows a marked increase in enzyme activity in oral cancer and suggests that adjacent normal tissue of unfavorable cases seems to change toward cancer. Moreover, it is conceivable to hypothesize that NNMT could represent a potential biomarker for early and non-invasive diagnosis of oral cancer.

Antiangiogenic, antitumoural and antimetastatic effects of two distamycin A derivatives with anti-HIV-1 Tat activity in a Kaposi's sarcoma-like murine model

The antiangiogenic, antitumoural and antimetastatic effects of two novel sulphonic derivatives of distamycin A, PNU145156E and PNU153429, were studied in a Kaposi’s sarcoma-like tumour model obtained by injecting nude mice with cells releasing extracellular HIV-Tat protein, derived from a tumour which developed in a BK virus/tat transgenic mouse. Both PNU145156E and PNU153429 were administered intraperitoneally every fourth day for three weeks at doses of 100 or 50 mg/kg of body weight respectively, starting one day after injecting the tumour cells. Both drugs delayed tumour growth in nude mice, preventing neovascularization induced by the Tat protein. PNU153429 also significantly reduced the number and size of spontaneous tumour metastases. Both effects on tumour growth and metastases were augmented by treating simultaneously nude mice with 7.5 mg/kg of body weight of minocycline given per os daily for four weeks starting four days after injecting the tumour cells. Neither acute nor chronic toxic side-effects were observed during the life span of treated nude mice. Due to their antiangiogenic and anti-Tat effects, these drugs are promising for the treatment of Kaposi’s sarcoma in AIDS patients.

Vibrational mapping of sinonasal lesions by Fourier transform infrared imaging spectroscopy

Abstract. Fourier transform infrared imaging (FTIRI) is a powerful tool for analyzing biochemical changes in tumoral tissues. The head and neck region is characterized by a great variety of lesions, with different degrees of malignancy, which are often difficult to diagnose. Schneiderian papillomas are sinonasal benign neoplasms arising from the Schneiderian mucosa; they can evolve into malignant tumoral lesions (squamous cell carcinoma). In addition, they can sometimes be confused with the more common inflammatory polyps. Therefore, an early and definitive diagnosis of this pathology is mandatory. Progressing in our research on the study of oral cavity lesions, 15 sections consisting of inflammatory sinonasal polyps, benign Schneiderian papillomas, and sinonasal undifferentiated carcinomas were analyzed using FTIRI. To allow a rigorous description of these pathologies and to gain objective diagnosis, the epithelial layer and the adjacent connective tissue of each section were separately investigated by following a multivariate analysis approach. According to the nature of the lesion, interesting modifications were detected in the average spectra of the different tissue components, above all in the lipid and protein patterns. Specific band-area ratios acting as spectral markers of the different pathologies were also highlighted.

Potential involvement of nicotinamide N-methyltransferase in the pathogenesis of metabolic syndrome.

BACKGROUND Metabolic syndrome is a complex disorder characterized by the presence of insulin resistance (IR), type 2 diabetes mellitus (T2DM), impaired glucose tolerance (IGT), or impaired fasting glucose (IFG), plus at least two of the following conditions--hypertension, hyperlipidemia, obesity, and microalbuminuria. Metabolic syndrome exposes patients to a greater risk of developing cardiovascular disease (CVD) and is often associated with elevated levels of homocysteine (Hcy). In the current work, we analyzed the expression of nicotinamide N-methyltransferase (NNMT). Because NNMT is involved in Hcy metabolism and participates in the regulation of the cellular and plasma levels of this compound, we explored the role played by the enzyme in metabolic syndrome. METHODS Real-time PCR, immunohistochemistry, western blot analysis, and catalytic activity assay were performed to evaluate NNMT expression levels in adipose tissue from 10 Wistar Ottawa Karlsburg W (WOKW) rats, which are an animal model for metabolic syndrome, and from 10 Dark Agouti (DA) rats as the disease-resistant control strain. RESULTS NNMT mRNA, protein, and activity levels were significantly higher in adipose tissue obtained from WOKW rats compared with those observed in adipose tissue of DA rats. CONCLUSION Data reported in this study represent the first evidence supporting the hypothesis that NNMT could play an important role in the pathogenesis of metabolic syndrome and could have a potential for the development of a targeted therapy.