However, when PARP is impaired, cells are noted to become exquisitely sensitive to DNA damaging agents such as radiotherapy  and . As a result, the clinical development of PARP inhibitors has followed two approaches: 1) combining PARP1/2 inhibition with DNA-damaging agents, such as radiation, to derive additional therapeutic benefit; and 2) targeting tumor
cells with pre-existing defects in double-strand DNA break repair, such as Brease Cancer (BRCA)-deficient cells, which are genetically predisposed to die when PARP activity is lost . ABT-888 is an orally available, small molecule inhibitor of PARP which has been shown to potentiate the effects of alkylators and radiotherapy in xenograft tumor models . Recognizing the therapeutic potential of PARP-1/2 inhibition in PDAC, we have investigated the addition
of veliparib to focused radiation in vitro and in vivo using a novel preclinical pancreatic cancer buy GSI-IX radiation research model  and . The PDAC cell line, MiaPaCa-2, stably transfected with the luciferase-aminoglycoside phosphotransferase Dapagliflozin concentration fusion gene under the control of the elongation factor-1α promoter, was kindly provided by Dr. Ralph Graeser, ProQinase GMBH, Freiburg, Germany. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, and 100 units/mL penicillin/streptomycin. Subconfluent cell monolayers were removed using Immune system 0.25% trypsin containing 1 mmol/L EDTA (Invitrogen) and passaged at a ratio of 1:3 or utilized for study. Cells were seeded in triplicate monolayer and treated with varying doses of ionizing radiation using a 137Cs irradiator (5 Gy/min; Mark I, Shepherd and Associates), ABT-888 (Selleck Chemicals, Houston, TX), or a combination of the two. All in
vitro studies were performed in triplicate. When cells were co-treated, ABT-888 was added to the cell suspension 30 minutes prior to irradiation and left until routine media change at 48 hours. Cell viability was determined by the ability to convert a redox dye (resazurin) into a fluorescent end product (resorufin) using the Cell Titer-Blue® Assay (Promega Corporation, Madison, WI) at varying time points after treatment. Treatment doses resulting in 10% (IC10), 20% (IC20) and 50% (IC50) cell death were calculated for ABT-888 and irradiation, respectively. ABT-888 dose enhancement factors were determined after co-treatment with varying irradiation doses. Levels of apoptosis were determined using a chemiluminescent caspase 3/7 assay (G8091, Promega Corporation, Madison, WI) 48 hours after treatment with ABT-888, radiation, or a combination thereof. PARP-1/2 inhibition was quantitated using an enzyme-linked immunosorbent assay for PAR protein (Trevigen, Gaithersburg, MD) after treatment with ABT-888, radiation, ABT-888 plus radiation, or no treatment. Total protein extracts were harvested 6 hours after treatment and PAR levels were determined by chemiluminescence.