Concetta Maria Messina

Differentiative pathway activated by 3-aminobenzamide, an inhibitor of PARP, in human osteosarcoma MG-63 cells.

This study describes the molecular mechanism by which treatment with 3‐AB, a potent inhibitor of PARP, allows human osteosarcoma MG‐63 cells to restrict growth and enter differentiation. Our findings show that in MG‐63 cells, aberrant gene expression keeps Rb protein constitutively inactivated through hyperphosphorylation and this promotes uncontrolled proliferation of the cells. After 3‐AB‐treatment, the poly(ADP‐ribosyl)ation of nuclear proteins markedly decreases and this results in an increase in both the hypophosphorylated active form of Rb and pRb/E2F complexes. These effects are accompanied by G1 arrest, downregulation of gene products required for proliferation (cyclin D1, β‐catenin, c‐Jun, c‐Myc and Id2) and upregulation of those implicated in the osteoblastic differentiation (p21/Waf1, osteopontin, osteocalcin, type I collagen, N‐cadherins and alkaline phosphatase). Our study suggests that use of PARP inhibitors may induce a remodeling of chromatin with the reprogramming of gene expression and the activation of differentiation.

Identification and expansion of human osteosarcoma-cancer-stem cells by long-term 3-aminobenzamide treatment

A novel cancer stem‐like cell line (3AB‐OS), expressing a number of pluripotent stem cell markers, was irreversibly selected from human osteosarcoma MG‐63 cells by long‐term treatment (100 days) with 3‐aminobenzamide (3AB). 3AB‐OS cells are a heterogeneous and stable cell population composed by three types of fibroblastoid cells, spindle‐shaped, polygonal‐shaped, and rounded‐shaped. With respect to MG‐63 cells, 3AB‐OS cells are extremely smaller, possess a much greater capacity to form spheres, a stronger self‐renewal ability and much higher levels of cell cycle markers which account for G1‐S/G2‐M phases progression. Differently from MG‐63 cells, 3AB‐OS cells can be reseeded unlimitedly without losing their proliferative potential. They show an ATP‐binding cassette transporter ABCG2‐dependent phenotype with high drug efflux capacity, and a strong positivity for CD133, marker for pluripotent stem cells, which are almost unmeasurable in MG‐63 cells. 3AB‐OS cells are much less committed to osteogenic and adipogenic differentiation than MG‐63 cells and highly express genes required for maintaining stem cell state (Oct3/4, hTERT, nucleostemin, Nanog) and for inhibiting apoptosis (HIF‐1α, FLIP‐L, Bcl‐2, XIAP, IAPs, and survivin). 3AB‐OS may be a novel tumor cell line useful for investigating the mechanisms by which stem cells enrichment may be induced in a tumor cell line. The identification of a subpopulation of cancer stem cells that drives tumorigenesis and chemoresistance in osteosarcoma may lead to prognosis and optimal therapy determination. Expression patterns of stem cell markers, especially CD133 and ABCG2, may indicate the undifferentiated state of osteosarcoma tumors, and may correlate with unfavorable prognosis in the clinical setting. J. Cell. Physiol. 219: 301–313, 2009.

Effect of sodium dodecyl sulfate (SDS) on stress response in the Mediterranean mussel (Mytilus Galloprovincialis): regulatory volume decrease (Rvd) and modulation of biochemical markers related to oxidative stress.

In this study the effects of an anionic surfactant, sodium dodecyl sulfate (SDS), are assessed on the Mediterranean mussel (Mytilus galloprovincialis), exposed for 18 days at a concentration ranging from 0.1 mg/l to 1 mg/l. The effects are monitored using biomarkers related to stress response, such as regulatory volume decrease (RVD), and to oxidative stress, such as reactive oxygen species (ROS), endogenous antioxidant systems and Hsp70 levels. The results demonstrate that cells from the digestive gland of M. galloprovincialis, exposed to SDS were not able to perform the RVD owing to osmotic stress. Further, SDS causes oxidative stress in treated organisms, as demonstrated by the increased ROS production, in comparison to the controls (p<0.05). Consequently, two enzymes involved in ROS scavenging, superoxide dismutase (SOD) and catalase (CAT) have higher activities and the proportion of oxidized glutathione (GSSG) is higher in hepatopancreas and mantle of treated animals, compared to untreated animals (p<0.05). Furthermore Hsp70 demonstrates an up-regulation in all the analyzed tissues of exposed animals, attesting the stress status induced by the surfactant with respect to the unexposed animals. The results highlight that SDS, under the tested concentrations, exerts a toxic effect in mussels in which the disruption of the osmotic balance follows the induction of oxidative stress.

Paclitaxel and beta-lapachone synergistically induce apoptosis in human retinoblastoma Y79 cells by downregulating the levels of phospho-Akt.

Paclitaxel (PTX) and beta‐lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 µM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p‐Akt levels and in ps166‐MDM2, the phosphorylated‐MDM2 form that enters the nucleus and induces p53 export and degradation. Treatment with wortmannin or transfection with a dominant negative form of Akt anticipated at 24 h the effects induced by PTX/LPC, suggesting a protective role against apoptosis played by Akt in Y79 cells. In line with these results, we demonstrated that Y79 cells contain constitutively active Akt, which forms a cytosolic complex with p53 and MDM2 driving p53 degradation. PTX/LPC treatment induced a weakness of Akt–MDM2–p53 complex and increased nuclear p53 levels. Our results suggest that phospho‐Akt lowering is at the root of the apoptotic action exerted by PTX/LPC combination and provide strong validation for a treatment approach that targets survival signals represented by phospho‐Akt and inhibitor of apoptosis proteins. J. Cell. Physiol. 222: 433–443, 2010.

Effects of dietary administration of fenugreek seeds, alone or in combination with probiotics, on growth performance parameters, humoral immune response and gene expression of gilthead seabream (Sparus aurata L.)

ABSTRACT The use of immunostimulants is considered a promising preventive practice that may help to maintain animal welfare and a healthy environment, while increasing production and providing higher profits. The purpose of this study was to evaluate the effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds, alone or combined with one of the following probiotic strains: Bacillus licheniformis (TSB27), Lactobacillus plantarum or Bacillus subtilis (B46). Gilthead seabream were fed a control or one of the supplemented diets for 3 weeks. The effects of these supplemented diets on growth performance parameters and the humoral immune response (natural haemolytic complement, peroxidase, total IgM levels, proteases and antiproteases activities) were evaluated after 2 and 3 weeks of feeding. Simultaneously, the expression levels of some immune‐relevant genes (igm, tcr‐&bgr;, csfr1 and bd) were measured in the head‐kidney. Interestingly, all probiotic supplemented diets increased seabream growth rates, especially the B. licheniformis supplemented diet. Generally, humoral immune parameters were enhanced by the dietary supplementation at the different time points measured. The results showed a significant increases in the immune parameters, principally in fish fed only fenugreek or fenugreek combined with B. subtilis. Furthermore, real time qPCR revealed that dietary supplementation significantly enhances the expression of immune‐associated genes in the head‐kidney, particularly igm gene expression. These results suggest that fenugreek alone or combined with one of the probiotic strains mentioned enhances the immune response of gilthead seabream, a species with one of the highest rates of production in marine aquaculture. HIGHLIGHTSInnate immune response was enhanced by the fenugreek dietary supplementation.Humoral immune parameters were stimulates mainly by fenugreek + B. subtilis diet.Supplemented diets induced an important up‐regulation of igm gene expression.Fenugreek and probiotic tested could be considered as a good aqua feed supplements.The use of immunostimulants is a promising alternative to maintain animal welfare.

Toward shrimp consumption without chemicals: Combined effects of freezing and modified atmosphere packaging (MAP) on some quality characteristics of Giant Red Shrimp (Aristaeomorpha foliacea) during storage.

The combined effects of freezing and modified atmosphere packaging (MAP) (100% N2 and 50% N2+50% CO2) on some quality characteristics of Giant Red Shrimp (GRS) (Aristaeomorpha foliacea) was studied during 12-month storage. In particular, the quality characteristics determined proximal and gas compositions, melanosis scores, pH, total volatile basic-nitrogen (TVB-N), thiobarbituric acid (TBA) as well as free amino acid (FAA). In addition, the emergent data were compared to those subject to vacuum packaging as well as conventional preservative method of sulphite treatment (SUL). Most determined qualities exhibited quantitative differences with storage. By comparisons, while pH and TVB-N statistically varied between treatments (P<0.05) and TBA that ranged between ∼0.15 and 0.30 mg MDA/kg appeared least at end of storage for 100% N2 treated-group, the latter having decreased melanosis scores showed such treatments with high promise to keep the colour of GRS sample hence, potential replacement for SUL group. By comparisons also, while some individual FAA values showed increases especially at the 100% N2-treated group, the total FAAs statistically differed with storage (P<0.05). The combination of freezing and MAP treatments as preservative treatment method shows high promise to influence some quality characteristics of GRS samples of this study.

Variations of lipid and apolipoprotein content in lipoproteins during fasting in European sea bass (Dicentrarchus labrax L.)

Lipoproteins are involved in transport of exogenous and endogenous lipids. For this reason, the lipoprotein pattern undergoes drastic variation during fasting. In D. labrax, observed variations concern, the relative content of the five lipoproteins and their composition, both in lipid and apolipoprotein content. These data indicate the presence of a cascade mechanism of maturation involving very-low-density lipoprotein, (VLDL), low-density lipoprotein (LDL)-1 and LDL-2. In addition, a metabolic correlation involves VLDL and high-density lipoprotein (HDL). VLDL are functionally comparable with mammalian chylomicrons; in fact, they disappear rapidly after feeding from the chromatographic pattern and are composed of almost 80% triglycerides. From the evolutionary point of view, HDL represent the most conserved lipoproteins and are involved in transportation of endogenous lipids, mobilized from accumulation tissues. Their apolipoprotein pattern is dominated by two apolipoproteins, apo A1 (26,000 Da) and apo A2 (12,000 Da).

Quality and antioxidant response of gilthead seabream (Sparus aurata L.) to dietary supplements of fenugreek (Trigonella foenum graecum) alone or combined with probiotic strains

Abstract The present study was conducted to determine the potential effect of the dietary intake of fenugreek (Trigonella foenum graecum) seeds alone or in combination with Bacillus licheniformis, Lactobacillus plantarum or B. subtilis on gilthead seabream quality and antioxidant response after 2 and 3 weeks of experimental feeding. The results showed that the supplements did not affect the percentage of the fatty acid profiles of muscle, demonstrating that all the additives tested can be administrated without any negative effect on biochemical composition and quality of gilthead seabream. The quantification of thiobarbituric acid reactive substances in muscle demonstrated the significant beneficial effect of the experimental diets compared with the control one. Besides, an increase in superoxide dismutase and catalase in liver was recorded after 3 weeks of administration of experimental diets. Furthermore, real time qPCR revealed that dietary supplementation with FEBS significantly enhances the expression of scavenging enzymes, such as cat and gr genes in the liver after 3 weeks. The findings suggest that the administration of fenugreek supplement alone or combined with probiotic strains could be considered as a good source of natural antioxidants and as a functional aquafeed ingredient for gilthead seabream. HighlightsAntioxidant status of seabream after dietary fenugreek + probiotics was evaluated.Supplemented diets did not affect the fatty acid profiles of the muscle.Experimental diets showed antioxidant properties (decreased the TBARS in muscle).SOD and CAT were enhanced after fed experimental diets for 3 weeks.Fenugreek + probiotics up‐regulated liver antioxidant‐related gene expression.

Anisakis pegreffii (Nematoda: Anisakidae) products modulate oxidative stress and apoptosis-related biomarkers in human cell lines

BackgroundIn countries with elevated prevalence of zoonotic anisakiasis and high awareness of this parasitosis, a considerable number of cases that associate Anisakis sp. (Nematoda, Anisakidae) and different bowel carcinomas have been described. Although neoplasia and embedded larvae were observed sharing the common site affected by chronic inflammation, no association between the nematode and malignancy were directly proved. Similarly, no data are available about the effect of secretory and excretory products of infecting larvae at the host’s cellular level, except in respect to allergenic interaction.MethodsTo test the mechanisms by which human non-immune cells respond to the larvae, we exposed the fibroblast cell line HS-68 to two Anisakis products (ES, excretory/secretory products; and EC, crude extract) and evaluated molecular markers related to stress response, oxidative stress, inflammation and apoptosis, such as p53, HSP70, TNF-α, c-jun and c-fos, employing cell viability assay, spectrophotometry, immunoblotting and qPCR.ResultsBoth Anisakis products led to increased production of reactive oxygen species (ROS), especially in EC-treated cells. While the ES treatment induces activation of kinases suggesting inflammation and cell proliferation (or inhibition of apoptosis), in EC-treated cells, other signaling pathways indicate the inhibition of apoptosis, marked by strong upregulation of Hsp70. Elevated induction of p53 in fibroblasts treated by both Anisakis products, suggests a significantly negative effect on the host DNA.ConclusionsThis study shows that in vitro cell response to Anisakis products can result in at least two different scenarios, which in both cases lead to inflammation and DNA damage. Although these preliminary results are far from proving a relationship between the parasite and cancer, they are the first to support the existence of conditions where such changes are feasible.

Tunisian tomato by-products, as a potential source of natural bioactive compounds

Abstract Consumption of tomato and tomato products is positively related to the reduction in cardiovascular disease and several types of cancer, thanks to the presence of natural compounds, such as antioxidants. Peels and seeds fractions of tomato, collected after industrial processing in Tunisian industries, were analysed for nutritional and antioxidants composition in perspective of its utilisation. Proximate composition, fatty acids profile, carotenoids, such as lycopene and beta-carotene, polyphenols contents, demonstrated the good potential of these residual products as a source of natural compounds, useful for food and nutraceuticals applications.