Oxadiazon affects the expression and activity of aldehyde dehydrogenase and acylphosphatase in human striatal precursor cells: A possible role in neurotoxicity.

Exposure to herbicides can induce long-term chronic adverse effects such as respiratory diseases, malignancies and neurodegenerative diseases. Oxadiazon, a pre-emergence or early post-emergence herbicide, despite its low acute toxicity, may induce liver cancer and may exert adverse effects on reproductive and on endocrine functions. Unlike other herbicides, there are no indications on neurotoxicity associated with long-term exposure to oxadiazon. Therefore, we have analyzed in primary neuronal precursor cells isolated from human striatal primordium the effects of non-cytotoxic doses of oxadiazon on neuronal cell differentiation and migration, and on the expression and activity of the mitochondrial aldehyde dehydrogenase 2 (ALDH2) and of the acylphosphatase (ACYP). ALDH2 activity protects neurons against neurotoxicity induced by toxic aldehydes during oxidative stress and plays a role in neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. ACYP is involved in ion transport, cell differentiation, programmed cell death and cancer, and increased levels of ACYP have been revealed in fibroblasts from patients affected by Alzheimer's disease. In this study we demonstrated that non-cytotoxic doses of oxadiazon were able to inhibit neuronal striatal cell migration and FGF2- and BDNF-dependent differentiation towards neuronal phenotype, and to inhibit the expression and activity of ALDH2 and to increase the expression and activity of ACYP2. In addition, we have provided evidence that in human primary neuronal precursor striatal cells the inhibitory effects of oxadiazon on cell migration and differentiation towards neuronal phenotype were achieved through modulation of ACYP2. Taken together, our findings reveal for the first time that oxadiazon could exert neurotoxic effects by impairing differentiative capabilities of primary neuronal cells and indicate that ALDH2 and ACYP2 are relevant molecular targets for the neurotoxic effects of oxadiazon, suggesting a potential role of this herbicide in the onset of neurodegenerative diseases.

Low doses of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, stimulate angiogenesis by regulating expression of urokinase type plasminogen activator and matrix metalloprotease 2.

BACKGROUND: Poly(ADP-Ribose) polymerase (PARP) activity has been demonstrated fundamental in many cellular processes, including DNA repair, cell proliferation and differentiation. In particular, PARP activity has been recently found to affect proliferation, migration, and tube formation of human umbilical vein endothelial cells. In recent times, PARP inhibitors have entered in clinical trials to potentiate cancer treatments by preventing DNA repair, but little is known about the effects performed by different drug concentrations on neoangiogenesis, an essential step in tumor growth. METHODS: Human umbilical vein endothelial cells were treated with 3 aminobenzamide (3ABA), a PARP inhibitor, and tested for several different cellular parameters. RESULTS: Here we present in vitro evidence that a low concentration of 3ABA (50 µM), stimulates angiogenesis by decreasing fibrinolytic activity, carried out by urokinase-type plasminogen activator (uPA), and by enhancing matrix metalloprotease-2 (MMP-2) gelatinolytic activity, in fibroblast growth factor-2-stimulated endothelial cells. These unbalanced pathways modify in vitro angiogenic steps, inhibiting chemoinvasion and stimulating tubulogenic activity. CONCLUSIONS: Our results suggest that the proangiogenic effect of low concentrations of 3ABA alerts on the efficacy of PARP inhibitors to potentiate anticancer therapy. Moreover, they indicate that endothelial chemoinvasion and tubulogenesis depend on distinct proteolytic pathways.

Differentiating and apoptotic dose-dependent effects in (-)-alpha-bisabolol-treated human endothelial cells.

The effect on angiogenesis of (-)-alpha-bisabolol [(-)-6-methyl-2-(4-methyl-3-cyclohexen-1-yl)-5-hepten-2-ol] (1), a widely distributed plant sesquiterpene alcohol, was investigated for the first time. Human endothelial cells treated with 1 were analyzed for their ability to differentiate and organize in microvessels and for their sensitivity to this compound in terms of cytotoxicity and cell growth inhibition. Within 24 h of the treatment with 5 microM 1, cells underwent massive death. Apoptosis induction was responsible for cytotoxicity triggered by 1 as revealed by the release of cytochrome c from the mitochondria, reduction of the Bcl-2/Bax ratio, and caspase 3 activation. At a lower, non-apoptotic concentration (0.25 microM), 1 showed a differentiating effect resulting in growth inhibition, invasiveness reduction, and tubule stabilization.

Inhibition of PARP activity by PJ-34 leads to growth impairment and cell death associated with aberrant mitotic pattern and nucleolar actin accumulation in M14 melanoma cell line.

The capability of PARP activity inhibitors to prevent DNA damage recovery suggested the use of these drugs as chemo- and radio-sensitisers for cancer therapy. Our research, carried out on cultured human M14 melanoma cells, was aimed to examine if PJ-34, a potent PARP activity inhibitor of second generation, was per se able to affect the viability of these cancer cells without any DNA damaging agents. Using time-lapse videomicroscopy, we evidenced that 10 microM PJ-34 treatment induced severe mitotic defects leading to dramatic reduction of cell proliferation and to cell death. PJ-34 cytotoxic effect was further confirmed by analysis of cell viability and clonogenic assay. Absence of canonic apoptosis markers allowed us to exclude this kind of cell death. No single and/or double stranded DNA damage was evidenced. Immunofluorescence analysis showed an aberrant mitotic scenario in several cells and subsequent multinucleation suggesting an atypical way for cells to die: the mitotic catastrophe. The detection of aberrant accumulation of polymerised actin inside the nucleolus was noteworthy. Taken together, our results demonstrate that, targeting PARP activity by PJ-34, cancer cell survival is affected independently of DNA damage repair. Two findings are remarkable: (a) cisplatin concentration can be reduced by three quarters if it is followed by treatment with 10 microM PJ-34 for 24 h to obtain the same cytotoxic effect; (b) effects dependent on PJ-34 treatment are reversible. Our data suggest that, to reduce the harm done to non-tumour cells during chemotherapy with cisplatin, the latter could be coupled with PJ-34 treatment.

Oxidative DNA damage repair and parp 1 and parp 2 expression in Epstein-Barr virus-immortalized B lymphocyte cells from young subjects, old subjects, and centenarians.

Oxidative DNA damage has been implicated in the aging process and in some of its features such as telomere shortening and replicative senescence. Poly(ADP-ribosyl)ation is involved in many molecular and cellular processes, including DNA damage detection and repair, chromatin modification, transcription, and cell death pathways. We decided to examine the behavior of poly(ADP-ribosyl)ation in centenarians, i.e., those subjects who represent the best example of longevity having reached a very advanced age avoiding the main age-associated diseases. In this study we investigated the relationship between DNA repair capacity and poly(ADP-ribose) polymerase activity in Epstein-Barr virus-immortalized B lymphocyte cell lines from subjects of three different groups of age, including centenarians. Our data show that cells from centenarians have characteristics typical of cells from young people both in their capability of priming the mechanism of repair after H(2)O(2) sublethal oxidative damage and in poly(ADP-ribosyl)ation capacity, while in cells from old subjects these phenomena are delayed or decreased. Moreover, cells from old subjects show a constitutive expression level of both parp 1 and parp 2 genes reduced by a half, together with a reduced presence of modified PARP 1 and other poly(ADP-ribosyl)ated chromatin proteins in comparison to cells from young subjects and centenarians. Our data support the hypothesis that this epigenetic modification is an important regulator of the aging process in humans and it appears to be rather preserved in healthy centenarians, the best example of successful aging.

Poly(ADP-ribosyl)ation, a molecular switch of transcription, shows an attractive relationship with urokinase expression.

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins that consists in the transfer of ADP-ribose units from NAD+ onto protein acceptors to form long and branched polymers. PARP activity is stimulated either by genotoxic stimuli or by environmental factors. The negative charged polymers alter functional activity of several proteins involved in genome stability, gene expression, cell proliferation and differentiation. Increasing evidence supports the view that PARP, for its crucial position in DNA repair and DNA transcription, influences cell survival not only during tissue injure, but also in environmental homeostasis modification. Therefore, it may be considered a molecular switch in the control of transcription, eventually leading to the choice of cell for life and death. This review summarizes the recent findings on PARP activity and special emphasis is given to its role in urokinase-type plasminogen activator upregulation.

FGF2-mediated upregulation of urokinase-type plasminogen activator expression requires a MAP-kinase dependent activation of poly(ADP-ribose) polymerase.

Poly(ADP-ribosyl)ation is a post-translational modification of protein occurring in the nucleus by poly(ADP-ribose) polymerase enzyme activity. The main role of poly(ADP-ribose) polymerase system as "nick sensor" and DNA breaks repair is based on its activation via DNA strand breaks. Furthermore, poly(ADP-ribose) polymerase modifies the binding to DNA of several transcriptional factors by poly(ADP-ribosyl)ation, thereby regulating also transcriptional gene expression. We have analyzed whether poly(ADP-ribose) polymerase activity is involved in basic fibroblast growth factor (FGF2)-mediated upregulation of urokinase-type plasminogen activator (uPA) mRNA. We demonstrated that specific inhibition of poly(ADP-ribose) polymerase activity via 3-aminobenzamide (3ABA) or NAD+ deprivation prevents FGF2-mediated uPA mRNA over-expression and cell-associated plasminogen activator (PA) production in GM7373 endothelial cell line. We verified that FGF2 stimulates poly(ADP-ribose) polymerase activity by a DNA strand breaks-independent manner which involves a mitogen-activated protein kinases (MAPK)-dependent pathway, as confirmed by using PD98059 inhibitor and anisomycin stimulation. Poly(ADP-ribose) polymerase involved in this mechanism is mainly the 60 kDa molecular mass isoform, that presents an increase in serine phosphorylation in the presence of FGF2.

Comparative levels of DNA breaks and sensitivity to oxidative stress in aged and senescent human fibroblasts: a distinctive pattern for centenarians.

Basal and H(2)O(2)-induced DNA breaks as well as DNA repair activity and efficacy of the antioxygenic system were determined in human dermal fibroblasts explanted from either (i) young donors and passaged serially to reach replicative senescence or (ii) young, old and centenarian donors and shortly propagated in culture. These fibroblasts have been employed as an in vitro and ex vivo model, respectively, to evaluate comparatively DNA integrity during senescence (increasing population doubling levels) and aging (increasing donor age). Constitutive levels of DNA total strand breaks, as determined by the alkaline extraction procedure, changed moderately among the different cell lines, which exhibited, however, significant differences in the amount of either single or double strand breaks. The former decreased along with both aging and senescence; the latter augmented during senescence while being virtually steady during aging. Moreover, fibroblasts from centenarians showed to be less sensitive to H(2)O(2)-induced DNA damage than other ex vivo fibroblasts. This feature could not account for either increased DNA repair activity or higher efficacy of the antioxygenic system and pointed, instead, to an intrinsic nuclear stability which might be typical of centenarian fibroblasts and potentially functional to longevity.