Cells were incubated in presence and absence of compounds. At the end of incubation time, cells were washed and resuspended (2 × 105 cells/ml) in Hank’s balanced salt solution (HBSS) cointaining 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA).

Following a further 20 min incubation at 37°C, DCF fluorescence was monitored by flow cytometry (FL1-H channel). In order to estimate the antioxidant potential of the compounds, control and teatred cells were exposed to 300 μM of the oxidant tert-bytylhydroperoxide (t-BOOH) for 30 min at 37°C before DCFH-DA loading. Topoisomerase I-Mediated DNA cleavage reactions APR-246 cost Human recombinant Top1 was purified from Baculovirus as previously described [23]. DNA cleavage reactions were performed using a 22-bp DNA oligonucleotide with a prominent Topoisomerase I cleavage site. Single-stranded oligonucleotide was labeled according to the manufacturers’ instructions by using terminal deoxynucleotidyltransferase (USB Corporation, Cleveland, HKI-272 mouse OHIO) that adds

a single labeled cordycepin molecule (γ-32P, 5000 Ci/mmol, PerkinElmer Life and Analytical check details Sciences, MA) to the 3′ terminus. Unincorporated nucleotides were removed by QIAquick Nucleotide Removal Kit (Qiagen, Hilden, Germany). The duplex DNA oligonucleotide was annealed by addition of an equal concentration of the complementary strand, heated to 95°C and slow cooled to room temperature. For the Toposomerase I cleavage reaction, DNA oligonucleotides were reacted for 20 min at 25°C with a 12 ng/mL solution

of human Topoisomerase I and the desired amount of drugs, in 10 Parvulin mM Tris–HCl pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA and 15 μg/mL bovine serum albumin. Reactions were stopped by adding 0.5% SDS and formamide containing 0.25% bromophenol blue and xylene cyanol, heated at 95°C for 5 min and chilled on ice. Reaction products were separated in 20% polyacrylamide denaturing sequencing gels. Dried gels were visualized using a B40 Storm phosphor imager (Amersham Biosciences, GE Healthcare, UK). Topoisomerase II-Mediated DNA cleavage reactions DNA was purchased from Invitrogen Corporation (Carlsbad, CA). It represents a portion of SV40 sequence, in particular from position 3449 to 3538, that contains prominent topoisomerase II cleavage sites [24]. DNA was purified on denaturing 20% polyacrylamide gel, recovered by soaking gel slices in water and then ethanol precipitated. Single-stranded DNA was 5′-labeled using T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) with [γ-32P]ATP (3000 μCi/mmol, PerkinElmer Life and Analytical Sciences, MA) according to the manufacturers’ instructions. Unincorporated nucleotides were removed by QIAquick Nucleotide Removal Kit (Qiagen, Hilden, Germany). The duplex DNA was annealed by addition of an equal concentration of the complementary strand, heating to 95°C and slow cooling to room temperature.