PMC

(A) Miapaca-2 and PANC-1 cells were pretreated with HS-173 (0-10 μM) for 6 h and then irradiated (4 Gy). After 24 h, cells were fixed and analyzed for apoptotic cell death by TUNEL assay. (B) Miapaca-2 cells were treated with HS-173 for 6 h and then irradiated (10 Gy). After 24 h, cell lysates were prepared and analyzed by Western blotting for cleaved PARP, cleaved caspase-3, and survivin. Data are expressed as means ± S.D. from three experiments (^*P < 0.05).

(A) Miapaca-2 cells were treated with HS-173 (0-10 μM) for 6 h before irradiation (10 Gy). After 24 h, cells were stained with PI and analyzed by flow cytometry. Quantitative PI staining data is presented as a percentage of the cell cycle distribution. (B) Miapaca-2 cells were treated with HS-173 for 6 h before irradiation (10 Gy). After 24 h, p-Cdc2 levels were assayed by Western blotting. Data are expressed as means ± S.D. from three experiments (^***P < 0.001).

(A) Miapaca-2 and PANC-1 cells were pretreated for 6 h with or without different concentrations of HS-173, and then exposed to radiation (10 Gy) for 30 min. p-DNA-PKcs, p-KAP1, and p-53BP1 levels were determined by Western blotting. (B) Miapaca-2 and PANC-1 cells were pretreated for 6 h with or without different concentrations of HS-173, then exposed to radiation (4 Gy) for 30 min. Cells were then stained for p-KAP1 and p-53BP1 (red) by immunofluorescence, and immunostaining was quantified densitometrically. Data are expressed as means ± S.D. from three experiments (^*P < 0.05, ^**P < 0.01 and ^***P < 0.001).

Miapaca-2 (A) and PANC-1 (B) pancreatic cancer cells were treated with different concentrations of HS-173 for 24 h and then irradiated with the indicated doses (0–6 Gy). After 2 h, media were changed and cells were processed for clonogenic survival assays at the end of experiments (10-14 days). Colonies were counted by eye, using a cut-off value of 50 viable cells per colony. Data are presented as means ± S.D. from triplicate experiments (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. control).

(A) Pancreatic cancer cells were irradiated with the indicated dose (5 and 10 Gy). In radiation condition, the high expression of p-AKT was observed in pancreatic cancer cells using Western blotting and immunoflorescence. (B) Miapaca-2 and PANC-1 cells were exposed to radiation (10 Gy) alone and/or together with different concentrations of HS-173 for 6 h. p-ATM and p-AKT levels were assessed by Western blotting. (C) Miapaca-2 and PANC-1 cells were pretreated with HS-173 for 6 h, irradiated (4 Gy) for 30 min, and then immunostained for γ-H2AX. γ-H2AX staining was quantified by analyzing the optical density of stained cells. Data are expressed as means ± S.D. from three experiments (^*P < 0.05 and ^**P < 0.01).

(A) Experiment schedule. Nude mice were intraperitoneally administered HS-173 (10 mg/kg, 5 times per week) and then irradiated (IR1: 2 Gy, twice a week for 3 weeks; IR2: 8 Gy, once). (B and C) Tumor size and body weight were measured every 2 days. After 34 days, tumors were excised and blood was collected for evaluation of ALT levels. A set of tumors excised at the end of the treatment period illustrates the marked reduction in tumor size with combination treatment. (D and E) Isolated pancreatic tumors were sectioned (8-μm) and stained for p-ATM, p-53BP1 and p-KAP1 by immunofluorescence and immunohistochemistry. Data are presented as means ± S.D. (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001).