ß-Catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids.

Cancer cells form three-dimensional (3D) multicellular aggregates (or spheroids) under non-adherent culture conditions. In ovarian cancer (OC), spheroids serve as a vehicle for cancer cell dissemination in the peritoneal cavity, protecting cells from environmental stress-induced anoikis. To identify new targetable molecules in OC spheroids, we investigated gene expression profiles and networks upregulated in 3D vs traditional monolayer culture conditions. We identified ALDH1A1, a cancer stem cell marker as being overexpressed in OC spheroids and directly connected to key elements of the ß-catenin pathway. ß-Catenin function and ALDH1A1 expression were increased in OC spheroids vs monolayers and in successive spheroid generations, suggesting that 3D aggregates are enriched in cells with stem cell characteristics. ß-Catenin knockdown decreased ALDH1A1 expression levels and ß-catenin co-immunoprecipitated with the ALDH1A1 promoter, suggesting that ALDH1A1 is a direct ß-catenin target. Both short interfering RNA-mediated ß-catenin knockdown and A37 ((ethyl-2-((4-oxo-3-(3-(pryrrolidin-1-yl)propyl)-3,4-dihydrobenzo [4,5]thioeno [3,2-d]pyrimidin-2-yl)thio)acetate)), a novel ALDH1A1 small-molecule enzymatic inhibitor described here for the first time, disrupted OC spheroid formation and cell viability (P<0.001). ß-Catenin knockdown blocked tumor growth and peritoneal metastasis in an OC xenograft model. These data strongly support the role of ß-catenin-regulated ALDH1A1 in the maintenance of OC spheroids and propose new ALDH1A1 inhibitors targeting this cell population.

Homocysteine-induced toxicity increases TG2 expression in Neuro2a cells.

High levels of homocysteine promote cell damage mainly through induction of oxidative stress, endoplasmic reticulum (ER) stress, and activation of pro-inflammatory factors. The effects of homocysteine were here examined in the continuously dividing neuroblastoma cell line Neuro2a. Cell treatment with homocysteine (100-500 microM) for 4 h increased ROS production while reducing cell viability in a dose-dependent manner. Cell exposure to 250 microM homocysteine was able to induce transglutaminase 2 up-regulation and increased in situ transglutaminase activity. These effects were prevented by the incubation with the transglutaminase activity inhibitor cystamine. Homocysteine also induced NF-kappaB activation that seemed associated with transglutaminase 2 up-regulation since the specific NF-kappaB inhibition by SN50 was able to reduce transglutaminase expression and activity levels. In the light of these observations, it may be postulated that TG2 up-regulation is involved in cell response to homocysteine-induced stress, in which NF-kappaB activation plays also a pivotal role.

Role of videofluorography swallow study in management of dysphagia in neurologically compromised patients.

PURPOSE: The aim of this study was to demonstrate the key role of the videofluorography swallow study (VFSS) in the management of patients with dysphagia and varying degrees of neurological deficit. MATERIALS AND METHODS: In 1 year (March 2004-March 2005) 47 patients with oropharyngeal dysphagia due to different types of neurological deficit and who required rehabilitation were studied. All patients underwent: (1) clinical history assessment, (2) speech therapy assessment and (3) VFSS using digital fluoroscopy (25 frames per second). Patients were divided according to the Waxman classification into seven levels of dysphagia, and the most suitable type of feeding was selected (normal diet, restricted diet, artificial nutrition). At discharge, the possibility of changing the dietary regimen followed in hospital was evaluated based on clinical progress, radiological follow-up and the degree of improvement obtained. RESULTS: VFSS confirmed aspiration in 21/47 (44%) patients, of whom four (8%) had not been suspected at clinical-speech therapy assessment. In 13/47 (28%) patients, VFSS identified changes at the oral (three patients) or pharyngeal stage (three patients) or both (seven patients) but with no signs of silent aspiration. In the remaining 13 (28%) patients, VFSS did not show any changes in swallow dynamics. On the basis of these data, together with the follow-up at the end of rehabilitation treatment, different nutritional strategies were adopted: artificial nutrition [percutaneous endoscopic gastrostomy (PEG) or nasogastric tube], a restricted-consistency diet or normal diet. CONCLUSIONS: Our experience shows that VFSS precisely classifies the degree of dysphagia that conditions the dietary management of each neurologically compromised patient.

Screening for C677T and A1298C MTHFR polymorphisms in patients with epilepsy and risk of hyperhomocysteinemia.

Hyperhomocysteinemia can result from decreased methylenetetrahydrofolate reductase (MTHFR) enzyme activity, owing to genetic polymorphisms andor inadequate folate intake. This study was aimed at investigating the prevalence of C677T and A1298C MTHFR polymorphisms, and their impact on hyperhomocysteinemia in 95 epileptic patients and 98 controls. Double gradient-denaturing gradient gel electrophoresis screening revealed that the frequency of T677 polymorphic allele was similar between cases and controls (46.3% vs 42.3%), whereas that of C1298 allele was significantly higher in patients (30.5% vs 19.4%, p < 0.05). Significant differences between the two groups were also found for the frequencies of genotypes AA1298 (46.3% in cases vs 67.3% in controls, p < 0.01) and AC1298 (46.3% in cases vs 26.6% in controls, p < 0.01). Other genotype frequencies did not show any statistically significant differences. Haplotype frequencies significantly differed between the two groups. The CT677/AC1298 diplotype was significantly more frequent in epileptic patients than in controls (32.6% vs 18.4%, p < 0.05). Patients treated with enzyme-inducing antiepileptic drugs, having this diplotype and concomitant low folate concentration (i.e., < 3.4 nmol/L), exhibited plasma homocysteine levels significantly higher than normal values (27.1 +/- 2.44 micromol/L, p < 0.001). This increase, however, was lower than that observed in folate-deficient patients with diplotype TT677/AA1298 (41.3 +/- 3.41 micromol/L, p < 0.001). Indeed, these two diplotypes could be regarded as risk factors for hyperhomocysteinemia. Conversely, we found that the CC677/AA1298 diplotype was significantly more frequent in controls (p < 0.01), suggesting a protective role. Our study suggests that both C677T and A1298C MTHFR polymorphisms should be examined when assessing genetic risk factors of hyperhomocysteinemia in epilepsy.