Treatment of FANCA Cells with Resveratrol and N-Acetylcysteine: A Comparative Study

et al. (2014) Treatment of FANCA Cells with Resveratrol and N-Acetylcysteine: A Comparative Study. PLoS ONE 9(8): e104857. doi:10.1371/journal.pone.0104857 Treatment of FANCA Cells with Resveratrol and N- Acetylcysteine: A Comparative Study Marta Columbaro 0 Silvia Ravera 0 Cristina Capanni 0 Isabella Panfoli 0 Paola Cuccarolo 0 Giorgia Stroppiana 0 Paolo Degan 0 Enrico Cappelli 0 Mauro Salvi, University of Padova, Italy 0 1 SC Laboratory of Musculoskeletal Cell Biology, IOR , Bologna , Italy , 2 DIFAR-Biochemistry Lab., University of Genova , Genova , Italy , 3 CNR-National Research Council of Italy, Institute of Molecular Genetics, Unit of Bologna-IOR , Bologna, Italy, 4 S. C. Mutagenesis , IRCCS AOU San Martino - IST (Istituto Nazionale per la Ricerca sul Cancro) , Genova , Italy , 5 Centro di Diagnostica Genetica e Biochimica delle Malattie Metaboliche, Istituto Giannina Gaslini , Genova , Italy , 6 Hematology Unit, Istituto Giannina Gaslini , Genova , Italy Fanconi anemia (FA) is a genetic disorder characterised by chromosome instability, cytokine ipersensibility, bone marrow failure and abnormal haematopoiesis associated with acute myelogenous leukemia. Recent reports are contributing to characterize the peculiar FA metabolism. Central to these considerations appears that cells from complementation group A (FANCA) display an altered red-ox metabolism. Consequently the possibility to improve FA phenotypical conditions with antioxidants is considered. We have characterized from the structural and biochemical point of view the response of FANCA lymphocytes to N-acetyl-cysteine (NAC) and resveratrol (RV). Surprisingly both NAC and RV failed to revert all the characteristic of FA phenotype and moreover their effects are not super imposable. Our data suggest that we must be aware of the biological effects coming from antioxidant treatment. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The authors have no funding or support to report. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Fanconi anemia (FA) is a genetic disorder characterised by chromosome instability and cytokine hypersensitivity. Bone marrow failure and abnormal haematopoiesis are associated with high frequency to clonal hematopoietic stem cells (HSC) expansion, acute myelogeneous leukemia via a mechanism involving genomic instability and inflammation [1]. We recently reported [2,3] different structural abnormalities in FANCA cells underlying an impaired mitochondrial functionality affecting the energy metabolism. A defective respiration at the mitochondrial oxidative phosphorylation complex I is associated with a reduced ATP production and altered ATP/AMP ratio. These defects are consistently associated with impaired oxygen metabolism [4]. Therefore the possibility to improve FA patients physiological state with antioxidants as therapy adjuvants appears promising. N-acetyl-cysteine (NAC) is a sulphydryl-group providing compound which acts as a precursor of reduced glutathione (GSH) and as direct scavenger of reactive oxygen species (ROS). Intracellular reduced GSH is often depleted as a consequence of increased oxidative stress and inflammation. Hence NAC can regulate the red-ox status in the cells interfering with several signalling pathways. NAC is widely used in clinical treatment [5] as support in treatment of diseases related to oxidative stress. Actually almost 250 studies with NAC are enlisted in the Clinical Trials governmental registry (www.clinicaltrials.gov). Resveratrol (RV) is a naturally-occurring polyphenol mainly found in grapes. A growing body of literature has demonstrated the beneficial effects of RV on age-related metabolic deterioration and its protective role in metabolic diseases. RV exerts its potent anticarcinogenic effect by inducing apoptosis and inhibiting tumor promoter-induced cell transformation [6]. RV protects against the deregulation of energy homeostasis, up-regulates eNOS and many cellular anti-oxidant enzymes, down regulates TNFa and NF-kB expression and inhibits NADPH oxidases [7]. Moreover, crystallographic studies showed that RV (and other related polyphenols) directly inhibits the rotary mechanism of mitochondrial FoF1-ATP synthase (ATP synthase) by binding to a site in c-subunit and hence its ATP synthetic activity [8]. More than 70 clinical trials with RV are actually listed in the National Institutes of Health website. In FA, the use of NAC and RV has already been proposed. Treatment with NAC, in association with Lipoic Acid (LA) [9] increased cellular viability as well as GSH and ATP contents, and reduced spontaneous and DEB-induced chromosomal instability in lymphocyte from FA patients. The protective abilities of NAC, RV and tempol were compared in the FANCD2 murine model [10]. NAC and RV partially corrected the abnormal cell cycle state of the HSP cells and helped maintaining them in a quiescent state. In turn tempol substantially delayed tumor onset apparently without a beneficial effect on hematopoiesis. Finally an antioxidant dietary formulate containing lysine, proline, ascorbic acid and green tea extracts, was successfully reported to inhibit in vitro and in vivo FANCA-associated head and neck squamous cell carcinoma [11]. Notwithstanding the potential interest concerning these results the still crucial and open question is that we do not know yet which molecular mechanisms and metabolic pathways are relevant in the FA pathological phenotype. Here we evaluate the biological effects of RV and NAC, two most promising antioxidants which act with different biochemical mechanisms. Materials & Methods Ethics statement Study approval was obtained from the Ethics Committee at the Gaslini Hospital, Genova, Italy (protocol Nu J5002 date: 24/9/ 2010). Informed written consent was obtained from the adult subjects and from parents, on the behalf of their children, involved in the study. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Cell culture and treatments FANCA primary fibroblast cell lines, isogenic FANCA primary fibroblasts corrected with S11FAIN [12] retrovirus and wild type (wt) cells were grown as monolayer at 37uC in RPMI supplemented with 10% fetal calf serum and antibiotics. All the cell lines between the 5th and 15th passage, were grown with the same density and conditions. During treatment we didnt observe significant changes in the cellular viability. FANCA and wt lymphoblast cell lines were grown at 37uC in RPMI supplemented with 10% fetal calf serum and antibiotics. Primary lymphocytes were isolated using Ficoll-Paque Plus and grown at 37uC in RPMI supplemented with 10% fetal calf serum, antibiotics and phytohemagglutinin (20 (...truncated)