P Salamone

Chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch by topical application of resveratrol complexed with 2-hydroxypropyl-β-cyclodextrin.

Oral squamous cell carcinoma (OSCC) develops slowly and it is usually preceded by identifiable oral preneoplastic lesions (OPLs): chemoprevention could be a promising approach. Resveratrol (RV) is a plant-based agent characterized by a strong in vitro antineoplastic action, but this effect has not been clinically confirmed owing to its metabolic inactivation. In order to circumvent this limitation and to improve RV efficacy, it was locally applied and complexed with a protective and solubilising vehicle (2-hydroxypropyl-beta-cyclodextrin, HPbetaCD). The experimentation was performed in vitro on 7,12-dimethylbenz[a]anthracene-induced hamster OSCC cell line (HCPC I) and in vivo in the related animal model, by comparison of two RV-HPbetaCD formulations (cream and mouthwash) and RV alone. Vehicles and RV-formulations were free from toxicity. Antiproliferative action of RV on HCPC I was concentration- and time-dependent, and was improved in HPbetaCD-formulations. In vivo, RV prevented OPL and OSCC appearance and growth. Here, too, HPbetaCD-formulations (mainly mouthwash) demonstrated the best chemopreventive effects in terms of lesions prevalence, multiplicity, dimension, and histological signs of malignancy. HPLC detection of RV corroborated that its action is concentration-correlated and is improved by its inclusion in HPbetaCDs. In summary, our study demonstrates that RV is effective in the chemoprevention of DMBA-induced oral carcinogenesis and when it is complexed with HPbetaCDs its efficacy is significantly improved.

ErbB2 Receptor Over-Expression Improves Post-Traumatic Peripheral Nerve Regeneration in Adult Mice

In a transgenic mice (BALB-neuT) over-expressing ErbB2 receptor, we investigated the adult mouse median nerve in physiological and pathological conditions. Results showed that, in physiological conditions, the grip function controlled by the median nerve in BALB-neuT mice was similar to wild-type (BALB/c). Stereological assessment of ErbB2-overexpressing intact nerves revealed no difference in number and size of myelinated fibers compared to wild-type mice. By contrast, after a nerve crush injury, the motor recovery was significantly faster in BALB-neuT compared to BALB/c mice. Moreover, stereological assessment revealed a significant higher number of regenerated myelinated fibers with a thinner axon and fiber diameter and myelin thickness in BALB-neuT mice. At day-2 post-injury, the level of the mRNAs coding for all the ErbB receptors and for the transmembrane (type III) Neuregulin 1 (NRG1) isoforms significantly decreased in both BALB/c and BALB-neuT mice, as shown by quantitative real time PCR. On the other hand, the level of the mRNAs coding for soluble NRG1 isoforms (type I/II, alpha and beta) increased at the same post-traumatic time point though, intriguingly, this response was significantly higher in BALB-neuT mice with respect to BALB/c mice. Altogether, these results suggest that constitutive ErbB2 receptor over-expression does not influence the physiological development of peripheral nerves, while it improves nerve regeneration following traumatic injury, possibly through the up-regulation of soluble NRG1 isoforms.

Injured cardiomyocytes promote dental pulp mesenchymal stem cell homing

BACKGROUND The heart is unable to regenerate its tissues after severe injuries. Stem cell therapy appears to be one of the most promising approaches, though preclinical results are hitherto contradictory and clinical trials scanty and/or limited to phase-I. The limited knowledge about stem cell early homing in infarcted cardiac tissues can concur to this scenario. METHODS The stem cell migration was assessed in in-vitro and ex-vivo models of heart ischemia, employing a rat dental pulp stem cell line (MUR-1) that shares the same ontogenic progenitors with portions of the heart, expresses markers typical of cardiac/vascular-like progenitors and is able to differentiate into cardiomyocytes in-vitro. RESULTS Here, we demonstrated that the MUR-1 can reach the injured cells/tissue and make contacts with the damaged cardiomyocytes, likely through Connexin 43, N-cadherin and von Willebrand Factor mediated cell-cell interactions, both in in-vitro and ex-vivo models. Furthermore, we found that SDF-1, FGF-2 and HGF, but not VEGF are involved as chemotactic factors in MUR-1 migration, notifying a similarity with neural crest cell behavior during the organogenesis of both the splanchnocranium and the heart. CONCLUSIONS Herein we found a similarity between what happens during the heart organogenesis and the early migration and homing of MUR-1 cells in ischemic models. GENERAL SIGNIFICANCE The comprehension of molecular aspects underlying the early phases of stem cell migration and interaction with damaged organ contributes to the future achievement of the coveted stem cell-mediated organ regeneration and function preservation in-vivo.

Potentiation of amyloid-β peptide neurotoxicity in human dental-pulp neuron-like cells by the membrane lipid peroxidation product 4-hydroxynonenal

Lipid peroxidation is generally considered as primarily implicated in the pathogenesis of Alzheimers disease (AD); one of its more reactive end products, 4-hydroxynonenal (HNE), has been shown to cause neuron dysfunction and degeneration. HNE production in the brain is stimulated by the amyloid-β peptide (Aβ), whose excessive accumulation in specific brain areas is a hallmark of AD. Conversely, Aβ production is up-regulated by this multifunctional aldehyde. Findings reported here point to the ability of HNE and Aβ to interact, with consequent potentiation of Aβs cytotoxicity as determined in vitro using neuron-like cells derived from human dental-pulp progenitor cells. Preincubation of cells with the aldehyde markedly up-regulated Aβ uptake and intracellular accumulation, by overexpressing two of the three components of the plasma membrane multireceptor complex CD36/CD47/β1-integrin: experimental and clinical data indicate that intraneuronal accumulation of Aβ is an early event possibly playing a primary role in AD pathogenesis. That HNE-mediated overexpression of CD36 and β1-integrin, which plays a key role in HNEs potentiating Aβ neurotoxicity, in terms of necrosis, was confirmed when this effect was prevented by specific antibodies against the two receptors.

Self-Renewal and Multipotency Coexist in a Long-Term Cultured Adult Rat Dental Pulp Stem Cell Line: An Exception to the Rule?

The stemness state is characterized by self-renewal and differentiation properties. However, stem cells are not able to preserve these characteristics in long-term culture because of the intrinsic fragility of their phenotype easily undergoing senescence or neoplastic transformation. Furthermore, although isolated from the same original tissue using similar protocols, adult stem cells can display dissimilar phenotypes and important cell clone/species contamination. Finally, the lack of a clear standardization contributes to complicate the comprehension about the stemness condition. In this context, cell lines displaying a particularly stable phenotype must be identified to define one or multiple benchmarks against which other stem cell lines could be reliably assessed. The present paper demonstrates that it is possible to isolate from the rat dental pulp a stem cell line (MUR-1) that does not display neoplastic transformation in long-term culture. MUR-1 cells stably express a broad range of stemness markers and are able to differentiate into adipogenic, osteogenic, chondrogenic, neurogenic, and cardiomyogenic lineages independently of the culture passages. Moreover, serial in vitro passages have not changed their immunophenotype, proliferation capacity, or differentiation potential. The uniqueness of these characteristics candidates MUR-1 as a model to reliably improve the understanding of the mechanisms governing the stem cell fate in the same as well as in other stem cell populations.

A DNA Vaccine against ERBB2 Impairs Chemical Carcinogenesis in Random-Bred Hamsters

Vaccines against oncoantigens halt early neoplastic lesions in several cancer-prone, genetically engineered mouse models, whereas their ability to prevent chemical carcinogenesis has not been explored. This is a significant issue, as exposure to chemical mutagens is responsible for a substantial percentage of cancers worldwide. Here, we show that the archetypal oncoantigen ERBB2 is transiently overexpressed in Syrian hamsters during the early stages of 7,12-dimethylbenz[α]anthracene (DMBA)-induced oral carcinogenesis. Repeated DNA vaccinations against ERBB2 significantly reduce the number, size, and severity of oral lesions in a manner directly proportional to the anti-ERBB2 antibody response. These results support the prospects of vaccines as a fresh strategy in the management of individuals at risk for exposure to defined carcinogenic agents. Cancer Prev Res; 4(7); 994–1001. ©2011 AACR.

Differentiation-inducing factor 1 enhances 5-fluorouracil action on oral cancer cells inhibiting E2F1 and Thymidylate Synthase mRNAs accumulation

PurposeDifferentiation-inducing factor-1 (DIF-1) is a morphogen originally identified in the amoebozoan Dictyostelium discoideum. In mammalian cells, it has been shown to activate GSK3β, which in turn is expected to reduce levels of β-catenin and cyclin D1, thus mediating DIF-1 antiproliferative properties. Since this could alter the expression and activity of E2F1 transcription factor and consequently those of the prognostic marker/chemotherapy target thymidylate synthase (TS), we evaluated (1) whether DIF-1 could effectively regulate these genes, (2) whether it could interfere with cell viability, and (3) whether DIF-1 activity could enhance the efficacy of the TS inhibitor 5-fluorouracil (5-FU).MethodsWe investigated the effects of DIF-1 in continuous human cell lines derived from two oral tumor histotypes (corresponding to an adenosquamous and a squamous carcinoma) and a gingival epithelium. We evaluated mRNA accumulation by means of quantitative real-time PCR and efficacy of drugs on cell viability by means of MTT assay.ResultsDIF-1 inhibited the accumulation of E2F1 mRNA and reduces TS mRNA levels in tumor cell lines, but did not alter mRNA levels in the gingival counterpart. As a result, it inhibited proliferation preferentially of tumor cell in time- and concentration-dependent manner. Moreover, it enhanced cytotoxic effects of 5-FU only in tumor cell, whereas reduced them in the gingival counterpart.ConclusionsThese findings suggest a tumor-specific action of DIF-1 on oral carcinoma cells. Thus, interfering with E2F1 and TS transcription, DIF-1 potentiates TS enzymatic inhibitors.

NADPH oxidase 2 (NOX2) enzyme activation in patients with chronic inflammatory demyelinating polyneuropathy

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired immunomediated condition affecting the peripheral nervous system where probably macrophages are the primary effector cells for demyelination. Reactive oxygen species (ROS), catalyzed by the NOX family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, can induce peroxidation and are potentially injurious to myelin. Our aim was to assess the activity of NOX2, an isoform of NOX, in a series of CIDP patients and to analyze the effect of intravenous immunoglobulin (IVIg) on NOX2.

NADPH oxidases 2 activation in patients with Parkinson's disease

Fig. 1. Mean fluorescence intensity (MFI) of oxidative burst in granulocytes (up) and monocytes (down) of PD patients and related controls after stimulation with bacterial stimulus (E. Coli). Neutrophils provide the first line of defense of the innate immune system by phagocytosing, killing, and digesting bacteria. Killing was previously believed to be accomplished by oxygen free radicals and other reactive oxygen species (ROS) generated by the NADPH oxidase in a process called “respiratory burst”. NADPH oxidase (NOX) enzymes catalyze the formation of ROS, which play a role in the development of different neurological disorders (ND), particularly whose generated by the phagocytic isoform NOX2 that is highly expressed in activated phagocytes including neutrophils and microglia [1], where it is responsible for the respiratory oxidative burst. Increased ROS has been observed in Amyotrophic Lateral Sclerosis (ALS), and a lower NOX2 activity showed a significant increase of survival in ALS patients [2]. There is also evidence that NOX are expressed and activated in patients with Parkinsons Disease (PD) and other ND, though the underlying mechanisms of NOX2mediated oxidative stress in PD pathogenesis are still unknown [3]. To elucidate the links between NOX2 and PD, the aim of this study was to evaluate the enzyme activity measuring oxidative burst of granulocytes and monocytes (ROS production) in fresh whole blood of patients in comparison with matched healthy controls using PhagoburstTM assay by flow cytometry, according to the manufacturers instructions. A total of 40 consecutive patients, 20 men and 20 women (64.7± 8.4 mean age and SD) with diagnosis of idiopathic PD and 40 healthy sexand age-matched control subjects (65.1± 8.6) were enrolled in the study. Patients were recruited at the Parkinsons andMovement Disorders Clinic, ‘Rita Levi Montalcini’ Department of Neurosciences, University of Turin, Italy. 18 subjects were inpatients, 22 outpatients. The study was approved by local Ethic Committee; both patients and controls provided the signed informed consent and personal data were treated according to the current Italian directives (Law No. 196 of 30 June 2003). Diagnosis of PD was made according to the UK Brain Bank criteria by a neurologist expert in movement disorder. The disease duration was 14.4 ± 6.3 years; 95% of patients (38/ 40) were in levodopa þ benserazide treatment. UPDRS of inpatients was 24± 8 (average of the differences of UPDRS OFF score versus UPDRS ON score calculated for each