Maria Claudia Lazzè

Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells.

Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation.

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Grape waste extract obtained by supercritical fluid extraction contains bioactive antioxidant molecules and induces antiproliferative effects in human colon adenocarcinoma cells.

Grape waste management is one of the main problems of winery industries, but, conversely, grape waste contains a high amount of polyphenols that might protect against human diseases related to oxidative stress, such as colorectal cancer. Therefore, the aim of this work was to investigate the antioxidant and antiproliferative activities of a grape waste extract obtained by supercritical fluid extraction. Because the beneficial effect of grape is related to its content of polyphenolic molecules, the extract was chemically characterized by high-performance liquid chromatography in order to assess its major bioactive components. The antioxidant activity of the grape extract was determined. The results showed that the grape extract presents a strong antiradical activity in the in vitro 2,2-diphenyl-1-picrylhydrazyl radical assay and protects against reactive oxygen species production in human colon adenocarcinoma cells (Caco-2). In contrast, the extract did not protect in the citronellal thermooxidation system and showed a weak protective action against lipid peroxidation in Caco-2 cells. The clonogenic assay and the cell cycle distribution analysis showed that the grape extract has a significant antiproliferative effect in a tumor cell line. These data indicate that grape extract is a promising product to be used as an anti-free radical agent and could exert a chemopreventive action.

Study of the effects of a new pyrazolecarboxamide: changes in mitochondria and induction of apoptosis.

Drug resistance of cancer cells is often correlated with the evasion of apoptosis, thus a major goal in cancer research is to search for compounds able to counteract cancer by promoting apoptosis. A variety of compounds with anticancer activity are characterised by the presence of the pyrazole as core nucleus. We synthesised a panel of pyrrolyl-pyrazole-carboxamides and we focused on the new compound RS 2780 (N-2-phenylethyl 1-(4-chlorophenyl)-3-methyl-5-pyrrolylpyrazole-4-carboxamide). The biological effects of RS 2780 on cell proliferation and viability were first evaluated on human HeLa cancer cells. As revealed by cell growth and viability experiments, a 24-h treatment of HeLa cells with increasing concentrations of RS 2780 (ranging from 0.1 to 100 microM) proved to inhibit cell proliferation and to affect cell viability. Notably, the new compound was effective also on colon carcinoma SW613-B3 cells, which are extremely resistant to most drugs, while it does not alter the proliferation of normal fibroblasts. We observed that RS 2780 interferes with the structural and functional properties of mitochondria, leading to the activation of the mitochondria-dependent apoptotic pathway. Apoptosis occurrence was supported by a number of morphological and biochemical hallmarks, including chromatin condensation, internucleosomal DNA fragmentation, PARP-1 cleavage and caspase activation. In conclusion, our results demonstrate for the first time the antiproliferative properties of the new compound RS 2780 on HeLa and SW613-B3 cancer cells and show that its effects on mitochondria lead to apoptosis.

Molecular Alterations in Fibroblasts Exposed to Helicobacter pylori Broth Culture Filtrate: A Potential Trigger of Autoimmunity?

Dear Editor, Gastric epithelial cells are the immediate and major target of Helicobacter pylori in vivo, yet even normal human fibroblasts are sensitive to H. pylori broth culture filtrate [1]. In the latter system, gene profiling evidenced a significant upregulation of genes involved both in the cell-cycle and structural machineries (Table 1, in white), but, beyond these modifications, which by themselves were not unexpected, an unusual gene set, typically expressed in neuronal cells, was also upregulated (Table 1, in grey). Among these genes, at least peripheral myelin protein 22 (PMP22), myelin associated glycorpotein (MAG), and glutamate decarboxylase 67-kDa isoform (GAD67), are known to act as self-antigens in autoimmune diseases [2,3] and we demonstrated by cytometry a significant increase in

Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria.

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.