A second part of our study was IDO inhibitor related to the well established observation that after UV-A irradiation, psoralens undergo photolysis with the formation of new species in solution, the so called photooxidation photoproducts (POPs). POPs also present some biological activity: in fact, some papers showed their

antileukemic and immunosuppressive effects, which led us to hypothesize their possible biological contribution in PUVA therapy [14] and [15]. Recently, we also isolated and reported the erythroid differentiation induction by a specific 5-methoxypsoralen photoproduct [16]. Thus, the effect of POPs was also evaluated on the expression of embryo-fetal globin genes in K562 cells by quantititative real-time reverse transcription polymerase-chain reaction assay (RT-qPCR). Psoralens and angelicins belong to the collection of the Sciences of Drug Department in Padova University [17], [18] and [19]. If not specified elsewhere, all chemicals, biological buffers and cellular media were purchased from Sigma–Aldrich. Two HPW 125 Philips lamps, mainly emitting at 365 nm, were used for irradiation

experiments. The spectral irradiance of the source was 4.0 mW cm−2 as measured at the sample level by a Cole-Parmer Instrument Company radiometer (Niles, IL, USA) equipped with a 365-CX sensor. tuclazepam The human leukemia

K562 cells were cultured in a humidified atmosphere of 5% CO2/air in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 units/mL penicillin and 100 mg/mL Selleckchem Lapatinib streptomycin. Suspensions of 30,000 K562 cells/mL in complete medium were seeded in individual wells of a 24-well tissue culture microtiter plate. The plates were incubated at 37 °C for 24 h prior to the experiments. Stock solutions of furocoumarin derivatives were prepared in methanol and then diluted with Hank’s balanced salt solution (HBSS pH 7.2; the concentration of methanol was always lower than 0.5%) for irradiation experiments. After medium removal, 1 mL of the drug solution was added to each well, incubated at 37 °C for 30 min and then irradiated (1 and 2 J/cm2, which correspond to 4 and 8 min of irradiation at 0.25 J/cm2). After irradiation, the solution was replaced with complete medium and the plates were incubated for 5–7 days. The medium was never changed during this period. Erythroid differentiation was determined by counting blue benzidine-positive cells after suspending the cells in a solution containing 0.2% benzidine in 10% H2O2 and 0.5 M glacial acetic acid [7]. Cell phototoxicity was assessed by the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] test 5 days after irradiation [20].