Inhibition of proliferation and invasion in 2D and 3D models by 2-methoxyestradiol in human melanoma cells.

Despite the recent advances in the clinical management of melanoma, there remains a need for new pharmacological approaches to treat this cancer. 2-methoxyestradiol (2ME) is a metabolite of estrogen that has shown anti-tumor effects in many cancer types. In this study we show that 2ME treatment leads to growth inhibition in melanoma cells, an effect associated with entry into senescence, decreased pRb and Cyclin B1 expression, increased p21/Cip1 expression and G2/M cell cycle arrest. 2ME treatment also inhibits melanoma cell growth in colony formation assay, including cell lines with acquired resistance to BRAF and BRAF+MEK inhibitors. We further show that 2ME is effective against melanoma with different BRAF and NRAS mutational status. Moreover, 2ME induced the retraction of cytoplasmic projections in a 3D spheroid model and significantly decreased cell proliferation in a 3D skin reconstruct model. Together our studies bring new insights into the mechanism of action of 2ME allowing melanoma targeted therapy to be further refined. Continued progress in this area is expected to lead to improved anti-cancer treatments and the development of new and more effective clinical analogues.

Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells.

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFß/SMAD (transforming growth factor-ß/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.

Serum amyloid A is a growth factor for 3T3-L1 adipocytes, inhibits differentiation and promotes insulin resistance.

BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [³H-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-³H]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9±0.54%) compared with the control cells (39.8±2.2%, (***) P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPARγ2 (peroxisome proliferator-activated receptor γ 2), C/EBPß (CCAAT/enhancer-binding protein ß) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-³H]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor α, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.

Efficacy of sodium hyaluronate in the management of chemical and radiation cystitis.

Onset of cystitis in patients receiving immuno-chemotherapeutic agents by intravesical instillation for non-muscle invasive transitional cell carcinoma of the bladder or after radiotherapy for prostatic cancer is frequent and problematic, since it responds poorly and slowly to the usual symptomatic treatments. This iatrogenic complication often means that cancer therapy has to be interrupted on account of the bladder pathology symptoms and of course this has further clinical implications. The symptoms resemble those of the urgency/frequency and painful bladder syndromes, so we tested the treatment used for these disorders to see whether it helped in this difficult clinical situation. This prospective study therefore enrolled 69 male consecutive patients, between 54 to 81 years of age, with iatrogenic acute cystitis; in 15 the symptoms had appeared after radiotherapy for prostatic cancer, in 24 after intravesical BCG, in 30 after instillation of Mitomycin C (with Synergo thermotherapy for 12 of them). All patients were given intravesical instillations of sodium hyaluronate, 40 mg/50 mL, weekly for from 8 to 24 weeks, depending on how the symptoms released. In the first four weeks dexamethasone 32 mg was mixed in as a "cocktail", on account of its prompt and effective topical antiinflammatory action and good mucosal penetration. Longer use of cortisone is contraindicated because of the high risk of sensitization and it provided no evidence of any ability to overcome the severe urinary disturbances with lasting effect. In order to allow patients with marked overactive bladder to keep these drugs within the bladder, we instilled lidocaine 2% 30 mL, 30 minutes before. Patients recorded their bladder capacity (BC) by filling a micturition diary. Pain was assessed using a Visual Analog Scale (VAS) from 0 to 10 for the chemical cystitis cases at the beginning and end of treatment. After only four weeks BC increased in all patients, and urgency and pain disappeared. Treatment was continued, however, for another four weeks, even in patients with total remission of their symptoms as we had seen earlier that if it was stopped too soon the symptoms could return. In the chemical cystitis group the VAS score dropped from an initial mean of 8.6 to 0.9 at the end of treatment (P<0.0001). Mean BC rose from 58.4 to 283.7 mL in the chemical cystitis cases (P<0.0001), and from 85 to 243.3 mL (P<0.0001) in the radiotherapy patients. Overall 67 patients (97%) reported complete relief of dysuria and pain. Two treatment failures were due to a reduced compliance to treatment by the patients themselves. No adverse reactions were observed related to the catheters or drugs used. Patients with non-invasive bladder tumors were able to restart their cancer therapy. For cystitis induced by intravescical immuno-chemotherapy or pelvic radiotherapy this approach appears to achieve an effective and rapid cure with no adverse reactions, allowing the conclusion of treatments for non-invasive transitional cell-bladder cancer. Patients with chemical cystitis responded a little better than those who had received radiotherapy. Subsequent urinary cytology and cystoscopy ruled out bladder cancer progression in these cases after temporary postponement of the oncological treatment. Intravescical sodium hyaluronate seems a valid and quick therapeutic solution for iatrogenic cystitis from chemo or radiotherapy. After review literature, this strategy does not appear to have been used before for this particular problem.

The problems associated with the monitoring of complex workplace radiation fields at European high-energy accelerators and thermonuclear fusion facilities.

The European Commission is funding within its Sixth Framework Programme a three-year project (2005-2007) called CONRAD, COordinated Network for RAdiation Dosimetry. The organisational framework for this project is provided by the European Radiation Dosimetry Group EURADOS. One task within the CONRAD project, Work Package 6 (WP6), was to provide a report outlining research needs and research activities within Europe to develop new and improved methods and techniques for the characterisation of complex radiation fields at workplaces around high-energy accelerators, but also at the next generation of thermonuclear fusion facilities. The paper provides an overview of the report, which will be available as CERN Yellow Report.

Spinal cord gliomas and hydrocephalus: utility of neuroendoscopy.

OBJECTIVE: The aim of this study is to report on the role of neuroendoscopy during the management of hydrocephalus that led to the diagnosis of intracranial tumoral dissemination and the subsequent finding of a spinal cord glioma. METHODS AND RESULTS: We present two children each with an intramedullary astrocytoma that presented initially with hydrocephalus without spinal cord symptoms. In both cases leptomeningeal gliomatous dissemination was asserted during routine endoscopy for the management of hydrocephalus. The diagnosis of a cervical and a lower thoracic intramedullary tumor was made soon after on magnetic resonance imaging. CONCLUSIONS: Spinal cord MRI with contrast should be considered initially in selected cases of hydrocephalus without evident diagnosis. The intraoperative diagnosis of gliomatous dissemination and secondary hydrocephalus due to unrecognized spinal cord gliomas was possible, in our experience, with the routine use of the neuroendoscope.

Urodynamic and clinical evidence of acute inhibitory effects of intravesical nociceptin/orphanin FQ on detrusor overactivity in humans: a pilot study.

PURPOSE: Management of neurogenic incontinence is complex and available treatments are not satisfactory. Nociceptin/orphanin FQ, a recently discovered neuropeptide, has been reported to inhibit the voiding reflex in the rat. These experimental results prompted us to investigate the urodynamic and clinical effects of intravesical instillation of nociceptin/orphanin FQ in humans. MATERIAL AND METHODS: Our study involved 5 normal subjects (group 1) with a mean age of 40.4 years (range 21 to 54) and 9 patients (group 2) 40.4 years (24 to 54). All patients in group 2 presented with detrusor hyperreflexia refractory to standard therapy. They were invited to undergo a filling cystometrogram with saline solution and after 30 minutes, a new one with a solution containing 1 microM. nociceptin/orphanin FQ. The urodynamic parameters that were recorded included bladder capacity, volume threshold for the appearance of detrusor hyperreflexia and maximum bladder pressure. Clinical and urodynamic followup was performed after 15 days. The data were statistically analyzed with 1-way analysis of variance followed by the Dunnett test for multiple comparison considered statistically significant with p <0.05. RESULTS: Intravesical instillation of 1 microM. nociceptin/orphanin FQ in group 1 did not produce significant functional changes. This infusion in group 2 produced a statistically significant increase in mean bladder capacity and volume threshold for the appearance of detrusor hyperreflexia from 164 plus or minus standard deviation (SD) 84 to 301 +/- 118 and 93 plus or minus SD 41 to 231 +/- 104 ml. (p <0.05, respectively). Mean maximum bladder pressure decreased from 79 plus or minus SD 25 to 54 +/- 44 cm. water but was not statistically significant (p = 0.19). After 15 days an absence of clinical improvement was noticed in group 2, and the urodynamic control did not show any significant changes compared to the values before nociceptin/orphanin FQ treatment. No severe symptomatic reactions were observed during infusion of 1 microM. nociceptin/orphanin FQ. CONCLUSIONS: Our results demonstrate that nociceptin/orphanin FQ is able to elicit a robust inhibitory effect on voiding reflex in group 2 but not 1. The ideal dosage, route of administration of nociceptin/orphanin FQ and treatment interval are not yet established.

Cross-linking chitosan-Fe(III), an oral phosphate binder: studies in vitro and in vivo.

The objective was to evaluate the in vitro and in vivo phosphate binding properties of cross-linked chitosan iron (III) (CH-Fe(III)-CL), a potential oral phosphate binder for treating hyperphosphatemia. At equilibrium, the in vitro phosphate binding of CH-Fe(III)-CL was 23.6 mg g(-1) for simulated gastrointestinal conditions. In hyperphosphatemic rats, CH-Fe(III)-CL was similar to iron sulfate in reducing serum phosphate by about 35%.

[Benign hemangiopericytoma of the bladder: description of a case and review of the literature]. / Emangiopericitoma benigno della vescica: descrizione di un caso e revisione della letteratura.

Hemangiopericytoma (HPC) of the viscera is very rare and only six of these tumors arising in the urinary bladder have been previously reported. The differentiation of HPC from other neoplasms with prominent vascular pattern may be a diagnostic problem. The difficulty is further enhanced by the lack of distinctive immunohistochemical features of this tumor. Here is presented a case of benign HPC occurring in the right lateral wall of the bladder of a 37-year-old woman. The mass is completely transurethrally resected. Histologically, the tumor is characterized by a monomorphic population of polygonal or spindle-shaped cells packed around branching vascular channels, with "staghorn" configuration. There are no foci of necrosis or hemorrhage. The mitotic count is 2 per 10 HPF. Immunohistochemically, the tumor cells react with anti-bodies against Vimentin. Cytokeratin, Actin HHF35, S-100, CD 34, Factor VIII-related antigen are not expressed within the lesion. Ultrastructurally, the tumor cells surround endothelium-lined vascular channels. There are pinocytotic vesicles on the cell borders. The cells are separated from one another by deposits of basal lamina-like material and collagen. On the basis of clinical, histological, immunohistochemical and ultrastructural observations, we believe that the case reported is a primary benign HPC of the bladder. There is no evidence of recurrence or metastasis 32 months later.

Lung carcinoma in 36 patients with human immunodeficiency virus infection. The Italian Cooperative Group on AIDS and Tumors.

BACKGROUND: The current study describes the clinicopathologic characteristics of 36 patients with lung carcinoma and human immunodeficiency virus (HIV) infection observed within the Italian Cooperative Group on AIDS and Tumors (GICAT). METHODS: Patients with lung carcinoma and HIV infection collected by the GICAT between 1986-1998 were evaluated retrospectively. As a control group, the authors analyzed 102 patients age < 60 years with lung carcinoma but without HIV infection who were seen at the CRO, National Cancer Institute, Aviano, Italy between 1995-1996. RESULTS: Patients with lung carcinoma and HIV infection were younger (38 years vs. 53 years) and previously smoked more cigarettes per day (40 vs. 20) than the control group. The main histologic subtype was adenocarcinoma. TNM Stage III-IV disease was observed in 53% of the patients. The median CD4 cell count was 150/mm(3). The median overall survival was significantly shorter in the patients with HIV compared with the control group (5 months vs. 10 months; P = 0.0001). CONCLUSIONS: The results of the current study demonstrate that lung carcinoma in the HIV setting affects mainly young individuals with a history of heavy tobacco smoking and a moderately advanced immunodeficiency status. Lung carcinoma is associated with a more adverse outcome in HIV patients and represents the cause of death in the majority of these patients.

Therapy of non-small-cell lung cancer (NSCLC) in patients with HIV infection. GICAT. Cooperative Group on AIDS and Tumors.

Incidence and mortality of AIDS patients have significantly declined in the developed countries due to the very active anti-HIV combination therapy available today. Because of the prolongation of the survival expectancy of these patients, other non-AIDS defining tumours have been recently reported in several cohort studies with increased frequency. We want to report the clinico-pathological features and the outcome of 39 patients with lung cancer and HIV infection, collected by the Italian Cooperative Group on AIDS and Tumors (GICAT) between 1986 and December 1997. As a control group, we decided to evaluate patients, less than 60 years of age, with lung cancer but without HIV infection seen at the CRO, Aviano, during 1995 and 1996. The median age of the study group patients was 38 years (range 28-58) and 90% of them were males. Sixty-nine percent of patients were intravenous drug users and HIV infection was asymtomatic in 41% of patients. NSCLC was observed in 78% of patients, SCLC in 13% and mesothelioma in 8%. Among NSCLC, adenocarcinoma was the most frequently observed histological subtype (48%). No differences were found as regards the stage of disease at diagnosis and the histologic subtype in comparison with the control group. The median overall survival was significantly shorter for patients with HIV infection when compared to that of the control group (5 months vs. 10 months, P < 0.0001). In conclusion, the outcome of patients with SNCLC and HIV infection seems worse than that of the general population, suggesting a synergistic and/or addictive adverse effect of HIV on the outcome of lung cancer.

Hodgkin's disease in HIV-infected individuals.

The clinical and pathologic characteristics of Hodgkin's disease as they are diagnosed in patients with HIV infection differ from those of the general population. In fact, a higher frequency of unfavorable histologic subtypes, advanced stage, and poor therapeutic outcome have been reported in the HIV setting. Complete response rate after chemotherapy ranges from approximately 45% to 70%, although the disease-free survival is very short, and the median overall survival is only 18 months. The survival of these patients may be improved by the optimal combination of antineoplastic and antiretroviral treatment with the support of hematologic growth factors. However, new strategies of therapeutic intervention must be explored.

Concomitant therapy with subcutaneous interleukin-2 and zidovudine plus didanosine in patients with early stage HIV infection.

A phase II study was performed to evaluate the feasibility and activity of subcutaneous (SC) interleukin-2 (IL-2) administration plus zidovudine (ZDV) and didanosine (ddI) in patients with early stage HIV infection. Between October 1995 and October 1996, 12 patients completed 6 cycles of the following scheduled therapy: ZDV plus ddI and SC self-administration of 6 mIU of IL-2 at days 1 to 5 and 8 to 12 of a 28-day cycle for a total of 6 cycles (24 weeks). After 6 cycles, patients received only ZDV plus ddI and they were observed up for an additional 24 weeks. Our schedule was well tolerated as an outpatient regimen and led to a significant elevation in CD4 count, which lasted for 24 weeks after the end of IL-2 therapy. Moreover, CD4/CD25, as well as CD4/CD45RO and CD4/CD45RA, cell levels were significantly increased at the end of the therapy and remained significantly elevated after 24 weeks. During the 6 cycles, HIV-associated viremia was significantly decreased and, accordingly, we observed a significant decline of proviral DNA in peripheral blood mononuclear cells (PBMCs). During follow-up, 10 of 12 treated patients continued to show levels of HIV-related viremia <500 copies/ml. Our results demonstrated that IL-2 and ZDV plus ddI is a well tolerated and effective therapy for patients with HIV in early stages of the disease.

[Malformations of the scrotum and its contents]. / Le malformazioni dello scroto e del suo contenuto.

To introduce we report the embryological origin of the male genitals and we present the endocrine control of the sex phenotypic differentiation and the most important features of the sexual differentiation abnormalities (ambiguous genitalia of newborn, gonadal dysgenesis, chromosome anomalies). Subsequently we report the classification of cryptorchidism and its correlation with hormonal and histological abnormalities. Furthermore we tried to expose the various different thoughts about testicular descent, anomalies associated with cryptorchidism and implications with malignant degeneration and infertility. Finally we describe the Magenta hospital experience from 1972 to 1998 reporting the surgical procedures for undescended testicle and for scrotal anomalies.

[Psychogenic urinary retention in childhood. A severe case treated by an integrated global approach]. / La ritenzione urinaria psicogena nel bambino. Descrizione di un caso grave trattato con approccio multidisciplinare.

Micturating dysfunctions with urinary retention and with diurnal and nocturnal enuresis in children sometimes have a psychogenic genesis. They can appear during the period of development of complete control of micturition. A late recognition of this condition makes the prognosis worse, since high pressure and infections in the urinary tract can cause end-stage renal failure. Here we describe a dramatic case of a 3 year-old boy affected by a psychogenic urine and faecal retention with recurrent pyelonephritis, that was favourably treated for five years by an integrated approach involving clinicians, psychologist, educational and social operators and adoptive parents.

[Complex urodynamic diagnosis of prostatic obstruction]. / Diagnostica urodinamica complessa dell'ostruzione cervico-prostatica.

Pressure/flow study is nowadays essential in the routine diagnostic workup of prostatic obstruction. It is the single most useful investigation showing evidence of bladder outlet obstruction. Therefore it should always be performed before any surgical treatment aimed to relieve the obstruction and it should always be referred to whenever the results of a medical treatment are judged. Lack of indication to pressure/flow study is limited to few clinical conditions such as complete urinary retention, large bladder diverticulum, high grade vesicoureteral reflux, bladder stone and/or urinary tract infection. The investigation needs much experience while is carrying out in order to avoid artifacts and then to read the pressure/flow data obtained. Results evaluation is still debatable. Nevertheless recent computerized programs allow to reduce artifacts and to obtain more reproducible and comparable data in order to make more reliable the indication to treatment or to judge its results.