Targeting nicotinamide N-methyltransferase decreased aggressiveness of osteosarcoma cells.

BACKGROUND: Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS. METHODS: Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively. RESULTS: Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti-cancer therapy.

Betacyanins enhance vitexin-2-O-xyloside mediated inhibition of proliferation of T24 bladder cancer cells.

Betacyanins (BC) were purified from beetroot (Beta vulgaris var. rubra L.) and tested, alone or in combination with vitexin-2-O-xyloside (XVX) from Beta vulgaris var. cicla L., for their ability to reduce the proliferation rate in T24 bladder cancer cells. Combination of BC and XVX exhibited a synergistic effect concerning the inhibition of proliferation in T24 cancer cells at 24 and 48 h but not after 72 h of incubation. The induction of apoptosis was evidenced by means of fluorescence activated cell sorting (FACS) analysis, as well as through the increase in caspase 3 and 8 activities. Using RTqPCR experiments, it was shown that the combination of XVX + BC was able to enhance the expression levels of pro-apoptotic BAX and downregulate anti-apoptotic BIRC5 (survivin), as well as pro-survival CTNNB1 (ß-catenin). The most evident effect of BC was the increase of the activity of caspase 8, leading to induction of extrinsic apoptosis. Moreover, XVX, BC and their combination showed no cytotoxic effect on normal human skin NCTC 2544 keratinocytes. These results demonstrated the efficacy and the mechanisms of the action of BC and XVX, extracted from edible plants, and suggested that a diet or a nutrition supplement, enriched with these bioactive molecules, could be used in the prevention of human bladder cancer.

Pokemon proto-oncogene in oral cancer: potential role in the early phase of tumorigenesis.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) represents about 90% of all oral neoplasms with a poor clinical prognosis. To improve survival of OSCC patients, it is fundamental to understand the basic molecular mechanisms characterizing oral carcinogenesis. Dysregulation of oncogenes and tumor suppressor genes seems to play a central role in tumorigenesis, including malignant transformation of the oral cavity. MATERIALS AND METHODS: We analyzed the expression levels of the pro-oncogenic transcription factor Pokemon through real-time PCR, Western blot and immunohistochemistry in tumor, and normal oral tissue samples obtained from 22 patients with OSCC. The relationship between tumor characteristics and the level of Pokemon intratumor expression was also analyzed. RESULTS: Pokemon was significantly downregulated in OSCC. In particular, both mRNA and protein levels (tumor vs normal tissue) inversely correlated with histological grading, suggesting its potential role as a prognostic factor for OSCC. Moreover, a significant inverse correlation was found between Pokemon protein expression levels (OSCC vs normal oral mucosa) and tumor size, supporting the hypothesis that Pokemon could play an important role in the early phase of tumor expansion. CONCLUSION: This work shows that reduced expression of Pokemon is a peculiar feature of OSCC. Additional studies may establish the effective role of Pokemon in oral tumorigenesis.

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.

House dust mites (Der p 1, Der f 1), cat (Fel d 1) and cockroach (Bla g 2) allergens in indoor work-places (offices and archives).

BACKGROUND: Exposure to indoor allergens has already been shown to occur in many public places, including workplaces, in several countries. Aim of this study was to measure the levels of house dust mites, cat and cockroach allergens in indoor workplaces (offices and archives) in Italy and to evaluate the possible relationships between allergen levels and building characteristics, type of ventilation, indoor relative humidity and temperature. METHODS: Der f 1 and Der p 1, Fel d 1, Bla g 2 were measured by ELISA in dust samples collected from floors or upholstered seats of 160 workplaces. RESULTS: Detectable Der p 1 levels were found in 86 (54%) workplaces Der f 1 in 87 (55%), Fel d 1 in 86 (54%) and Bla g 2 in 3 (1.9%). Der p 1 allergen concentrations expressed per weight were higher than the proposed sensitization thresholds in 7 samples, Der f 1 in 5, Fel d 1 in 6 and Bla g 2 in 3. The highest allergen levels were detected in samples from upholstered seats. A significant correlation was found between Der f 1 level on floors, expressed per surface, and indoor temperature (r = 0.39; P < 0.01). CONCLUSIONS: In our study we found that upholstered seats in workplaces in Italy may constitute a significant reservoir both of house dust mites and cat allergens. Exposure to these allergens in workplaces may represent a risk factor for elicitation of symptoms and/or induction/maintenance of inflammation in allergic individuals and might also constitute a risk factor for sensitization.

Increased CD69 expression on peripheral blood eosinophils after specific inhalation challenge.

BACKGROUND: CD69 is a molecule expressed on human eosinophils after cytokine-activation. Different studies have described the eosinophil activation, evaluated by CD69 expression, at the site of an allergic inflammation. In this study we evaluated the expression of CD69 on peripheral blood eosinophils after a specific inhalation challenge (SIC), in order to better define the state of activation of peripheral blood eosinophils after exposure to sensitizers. METHODS: CD69 expression was evaluated by flow cytometry in nine asthmatic patients before and after a positive SIC with high or low molecular weight agents (pollens, house dust mites, Penicillia, isocyanates) and in 11 asthmatic patients who underwent an inhalation challenge with placebo. CD69 expression was evaluated at baseline, 120 min, and 240 min after the SIC or the placebo. RESULTS: Baseline (before challenge) CD69 expression was comparable between the group of SIC positive patients and the placebo group. CD69 expression on peripheral eosinophils significantly increased 240 min after the challenge in positive SIC patients compared to placebo. In patients with a positive SIC the percentage of peripheral blood eosinophils significantly decreased at 120 and 240 min after the inhalation challenge with respect to the baseline. CONCLUSION: CD69 expression on peripheral blood eosinophils is significantly increased in asthmatic patients after exposure to the sensitizing agent. These data show that the effects of a bronchial stimulation are also detectable on peripheral blood eosinophils.

[Sampling of allergens in dust deposited in the workplace]. / Il campionamento degli allergeni nelle polveri depositate nei luoghi di lavoro.

Some workplaces share with domestic dwellings many characteristics favouring house dust mite growth. Moreover it has recently been shown that pets owners can bring allergens to public places with their clothes. So it is possible that significant exposure to indoor allergens can occur outside homes, at the workplace. The recent availability of immunoassays with monoclonal antibodies for indoor allergens has enabled many investigators to quantify exposure to such allergens in epidemiological studies. Analysis of allergens in settled dust is a simple method of quantification exposure to indoor allergens. The concentrations of indoor allergens in public places have already been investigated and high levels of indoor allergens have been reported. A study performed by our group in offices (banks and media) in different regions of Italy has also shown significant levels of indoor allergens. Thus, evaluating exposure to indoor allergens at the workplace is critical to evaluate risk factors for sensitization and elicitation of symptoms in sensitized subjects and such data help in addressing correctly the problem of reducing exposure levels.

Levels of house-dust-mite allergen in homes of nonallergic people in Pavia, Italy.

BACKGROUND: HDM distribution varies between geographic areas and may be affected by housing characteristics. We quantified Der p 1 and Der f 1 and assessed the relationships between their levels and housing characteristics in homes of nonallergic subjects. METHODS: Der f 1 and Der p 1 were measured by ELISA in dust samples from living-room floors and mattresses of 44 homes of nonallergic subjects in Pavia. Information about household characteristics was obtained by questionnaire. RESULTS: Der p 1 and Der f 1 concentrations (microg/g dust, median) were 0.34 and 7.8 on mattresses, and 0.15 and 0.83 on living-room floors. Higher Der f 1 levels on mattresses were associated with synthetic pillows (P<0.05), and (only when expressed as microg/m2) with bedding washing temperature of < or = 60 degrees C (P<0.05). Der f 1 levels were higher on the living-room floors of homes located on lower (< or =first) floors (P<0.05). Good correlations were found between Der p 1 and Der f 1 expressed as microg/g dust and microg/m2 on both mattresses and living-room floors. CONCLUSIONS: In homes of nonallergic subjects in northern Italy, Der f 1 exceeded the threshold for sensitization in a high proportion of mattress samples.

Human papillomavirus DNA in cervical intraepithelial neoplasia detected by in situ hybridisation.

Human papillomavirus (HPV) infection was investigated by in situ hybridisation in histological sections from 38 women with abnormal Papanicolaou smears. 13 patients had condylomatous lesions without atypia, 15 cervical intraepithelial neoplasia (CIN) I, 4 CIN II, 3 CIN III and 2 carcinoma in situ (CIS). HPV DNA was detected in 29 cases (78%) (1 specimen was technically inadequate). HPV 16 and 18, and 31, 33 and 35 were both present (67%) in CIN III. HPV 6 and 11 were more frequent in CIN I (56%) and in condylomatous lesions (38%). 31% of the condylomatous lesions without atypia contained HPV 31, 33, and 35 and 31% of those with CIN I were infected with HPV 16 and 18. These data confirm the frequent association of HPV infection with cervical cancer and CIN, and indicate that in situ hybridisation can identify patients with specific types of HPV infection at risk for cervical cancer.

[Dyspepsia and Helicobacter pylori infection. A study of a population of workers]. / Dispepsia ed infezione da Helicobacter pylori. Studio su una popolazione di lavoratori.

We studied 102 workers in a mechanical factory. All subjects were tested by a questionnaire screening dyspeptic symptoms in the last three months, their frequency, absenteeism period, spontaneous drugs intake and a consultant gastroenterologist request. All subjects were tested for specific IgG against Helicobacter pylori with immunoassay Elisa. Dyspepsia was present in 61% of cases. Positivity for Helicobacter pylori infection was in 76%, absenteeism in 82%, consultant request in 21% of dyspeptic workers. Our results suggest, according to literature, the high frequency of Helicobacter pylori infection in dyspeptic patients: furthermore dyspepsia should be considered as an important cause of absenteeism.

[Randomized study of the use of synthetic diosmin in premenstrual and vascular dysplastic mastodynia]. / Studio randomizzato sull'impiego della diosmina sintetica nella mastodinia displasica premestruale e vascolare.

The antalgic effect of synthetic diosmin in 120 patients suffering from vascular and premenstrual mastodynia is studied compared to the same number of non-treated cases. The therapy has been carried out for one consecutive year. For the same period the course of the painful symptomatology, both in the cases treated and in the controls, has been checked. For the treated cases a significant decrease of the painful symptomatology has been obtained and this decrease was maintained.

Ultrastructure of fetal human gonad before sexual differentiation and during early testicular and ovarian development.

The development of the human gonad was investigated by light and transmission electron microscopy in 20 embryos and fetuses between 4.5 and 11.5 weeks of gestation, i.e. during the stages of sex-indifferent gonad, initial testicular and ovarian development. The gonadal blastema in 4.5-week-old embryos appeared formed by poorly differentiated somatic mesothelial cells, and by specialized germ cells (PGCs) with signs of ameboidism, cellular structures suggesting active protein biosynthesis and mitotic activity. The sexual differentiation of the gonads was clearly observed in 7-week-old embryos and involved at the same time the testis and the ovary. The former contained seminiferous cords formed by palisades of poorly differentiated Sertoli cells, which were segregated from the mesothelium by a rudimentary albuginea. The interstitial tissue at this age contained mesenchymal cells. Between 8 and 11.5 weeks of gestation, there was a synchronous cytodifferentiation of both Sertoli and Leydig cells. The latter acquired features of steroidogenic elements. The ovaries of 7-week-old fetuses contained packed ovigerous cords formed by somatic and germ cells (oogonia). The former embraced the oogonia with thin overlapping cytoplasm projections, and acquired features similar to those of cells in primary follicles, already at this early fetal age. At the same time the sexual differentiation of the gonads involved somatic and germ cells. In the female, the oogonia continued to show the main features they had during migration and colonization, including a high mitotic rate, signs of ameboidism and a developed apparatus for protein synthesis. On the contrary Gonocytes enclosed in the seminiferous cords progressively entered a quiescent phase characterized by a reduced mitotic rate, a decrease of endoplasmic reticulum and nucleolar complexity. The chronological relationship between the cytodifferentiation of Sertoli and Leydig cells, and changes of germ cells, suggest that somatic components of the testis may contribute to a male type of differentiation of germ cells from the very beginning of sexual differentiation.

Evaluation of tumor and trophoblastic marker concentration in breast cyst fluid.

In our study we have examined 314 samples of cyst fluid taken from women suffering from fibrocystic breast disease (gross cystic disease). We have subdivided the cyst fluid with respect to epithelial coating and we have related trophoblastic protein content of the cyst fluid with age, seriousness of illness, and cytology of epithelial lining. We have performed RIA analysis of the trophoblastic proteins betahCG, beta1-SP-1, and alphahCG and in a smaller (n=84) group of specimens we have also tested for CEA, TPA, and ferritin. Trophoblastic proteins were positive in cystic fluids but the biological meaning of this is not known and the values are not related to clinical manifestations, except in a group of patients with apocrine metaplasia in which we tried to find a relationship between fertile age and increased betahCG. This finding presumably has a prognostic meaning that can be further understood by epidemiologic studies (of dietary intake and evaluation of lipid metabolites) and by information about inflammatory state of cystic fluid.

[Contraception and Gardnerella vaginalis. First study of the Italian population]. / Contraccezione e Gardnerella vaginalis. Prima indagine sulla popolazione Italiana.

The association between contraception and Gardnerella vaginalis was studied in the population of L'Aquila. This research was conducted in the cytocolposcopic Center of the Obstetrics and Gynaecology Clinic, and the Department of Biotechnology and Biometry at L'Aquila University on 1212 females. Of the examined patients, 178 (14.7%) employed a prophylactic method and of these 104 (58.4%) used barrier-systems, while 74 (41.6%) used estroprogestins. Gardnerella vaginalis was isolated in 63 females (6.1%) that used no contraceptive and 19 (10.7%) that employed one.

[Cervical maturation. Physiologic aspects and pharmacologic implications]. / La maturazione cervicale. Aspetti fisiologici ed attualità farmacologiche.

The latest theories on cervical maturation and the techniques of pharmacological induction of labour are summarised. The role of the prostaglandins is particularly stressed both from the physiological viewpoint and as drugs of choice for effective pharmacological induction of uterine cervix. Finally, the personal clinical protocol for the induction and management of labour is reviewed.

PCB and other organochlorine compounds in blood of women with or without miscarriage: a hypothesis of correlation.

Polychlorobiphenyls (PCBs) have been shown to affect reproduction in experimental animals and in human beings following intoxication. The concentrations of PCBs and other organochlorine compounds were determined in blood samples from 120 women hospitalized for miscarriages and 120 full-term pregnancy controls. The average PCB hematic level (reported as Fenclor 54) was higher in women with miscarriages than in control women. No differences were detected as far as the hematic concentrations of hexachlorobenzene and DDT compounds were concerned. The reproductive history of each woman was assessed together with confounding variables on the phenomenon of miscarriage and the factors regarding environmental exposure and food intake. Food consumption did not indicate diet as the main source of PCB intake.