The serine/threonine protein kinase ATM signals to cell cycle and DNA repair components by phosphorylating downstream targets such as p53, CHK2, NBS1, and BRCA1. Mutation of ATM occurs in the human autosomal recessive disorder ataxia-telangiectasia, which is characterized by hypersensitivity to ionizing radiation and a failure of cells to arrest the cell cycle after the induction of DNA double-strand breaks. It has thus been proposed that ATM inhibition would cause cellular radio-and chemosensitization. Through screening a small molecule compound library developed for the phosphatidylinositol 3-kinase-like kinase family, we identified an ATP-competitive inhibitor, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), that inhibits ATM with an IC 50 of 13 nmol/L and a Ki of 2.2 nmol/L. KU-55933 shows specificity with respect to inhibition of other phosphatidylinositol 3-kinase-like kinases. Cellular inhibition of ATM by KU-55933 was demonstrated by the ablation of ionizing radiation-dependent phosphorylation of a range of ATM targets, including p53, ␥H2AX, NBS1, and SMC1. KU-55933 did not show inhibition of UV light DNA damage induced cellular phosphorylation events. Exposure of cells to KU-55933 resulted in a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA double-strand break-inducing chemotherapeutic agents, etoposide, doxorubicin, and camptothecin. Inhibition of ATM by KU-55933 also caused a loss of ionizing radiation-induced cell cycle arrest. By contrast, KU-55933 did not potentiate the cytotoxic effects of ionizing radiation on ataxia-telangiectasia cells, nor did it affect their cell cycle profile after DNA damage. We conclude that KU-55933 is a novel, specific, and potent inhibitor of the ATM kinase.

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PK CS (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by ;H2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation-induced and etoposide-induced DSB. NU7441 appreciably increased G 2 -M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use.

BackgroundPeptidylarginine deiminase type 4 (PAD4/PADI4) post-translationally converts peptidylarginine to citrulline. Recent studies suggest that PADI4 represses expression of p53-regulated genes via citrullination of histones at gene promoters.MethodsExpression of PADI4 was investigated in various tumors and non-tumor tissues (n = 1673) as well as in A549, SKOV3 and U937 tumor cell lines by immunohistochemistry, real-time PCR, and western blot. Levels of PADI4 and citrullinated antithrombin (cAT) were investigated in the blood of patients with various tumors by ELISA (n = 1121).ResultsImmunohistochemistry detected significant PADI4 expression in various malignancies including breast carcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cancer cells, colorectal adenocarcinomas, renal cancer cells, ovarian adenocarcinomas, endometrial carcinomas, uterine adenocarcinomas, bladder carcinomas, chondromas, as well as other metastatic carcinomas. However, PADI4 expression was not observed in benign leiomyomas of stomach, uterine myomas, endometrial hyperplasias, cervical polyps, teratomas, hydatidiform moles, trophoblastic cell hyperplasias, hyroid adenomas, hemangiomas, lymph hyperplasias, schwannomas, neurofibromas, lipomas, and cavernous hemangiomas of the liver. Additionally, PADI4 expression was not detected in non-tumor tissues including cholecystitis, cervicitis and synovitis of osteoarthritis, except in certain acutely inflamed tissues such as in gastritis and appendicitis. Quantitative PCR and western blot analysis showed higher PADI4 expression in gastric adenocarcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cell cancers and breast cancers (n = 5 for each disease) than in the surrounding healthy tissues. Furthermore, western blot analysis detected PADI4 expression in cultured tumor cell lines. ELISA detected increased PADI4 and cAT levels in the blood of patients with various malignant tumors compared to those in patients with chronic inflammation and benign tumors. This was consistent with immunohistochemical results. Additionally, PADI4 and cAT levels were significantly associated with higher levels of known tumor markers.ConclusionOur results suggest that PADI4 expression is increased in the blood and tissues of many malignant tumors, a finding useful for further understanding of tumorigenesis.

BackgroundAutonomic nervous system dysfunction is implicated in the etiopathogenesis of inflammatory bowel diseases (IBD). Therapies that increase cardiovagal activity, such as Mind-Body interventions, are currently confirmed to be effective in clinical trials in IBD. However, a poor understanding of pathophysiological mechanisms limits the popularization of therapies in clinical practice. The aim of the present study was to explore the mechanisms of these therapies against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats using a chronic vagus nerve stimulation model in vivo, as well as the lipopolysaccharide (LPS)-induced inflammatory response in human epithelial colorectal adenocarcinoma cells (Caco-2) by acetylcholine in vitro.Methods and ResultsColitis was induced in rats with rectal instillation of TNBS, and the effect of chronic VNS (0.25 mA, 20 Hz, 500 ms) on colonic inflammation was evaluated. Inflammatory responses were assessed by disease activity index (DAI), histological scores, myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS), TNF-α and IL-6 production. The expression of Mitogen-activated protein kinases (MAPK) family members, IκB-α, and nuclear NF-κB p65 were studied by immunoblotting. Heart rate variability (HRV) analysis was also applied to assess the sympathetic-vagal balance. DAI, histological scores, MPO activity, iNOS, TNF-α and IL-6 levels were significantly decreased by chronic VNS. Moreover, both VNS and acetylcholine reduced the phosphorylation of MAPKs and prevented the nuclear translocation of NF-κB p65. Methyllycaconitine (MLA) only reversed the inhibitory effect on p-ERK and intranuclear NF-κB p65 expression by ACh in vitro, no significant change was observed in the expression of p-p38 MAPK or p-JNK by MLA.ConclusionVagal activity modification contributes to the beneficial effects of the cholinergic anti-inflammatory pathway in IBD-related inflamed colonic mucosa based on the activation of MAPKs and nuclear translocation of NF-κB. Our work may provide key pathophysiological mechanistic evidence for novel therapeutic strategies that increase the cardiovagal activity in IBD patients.

Ataxia telangiectasia mutated (ATM) kinase signals DNA double-strand breaks (DSB) to cell-cycle arrest via p53 and DNA repair. ATM-defective cells are sensitive to DSB-inducing agents, making ATM an attractive target for anticancer chemo-and radiosensitization. KU59403 is an ATM inhibitor with the potency, selectivity, and solubility for advanced preclinical evaluation. KU59403 was not cytotoxic to human cancer cell lines (SW620, LoVo, HCT116, and MDA-MB-231) per se but significantly increased the cytotoxicity of topoisomerase I and II poisons: camptothecin, etoposide, and doxorubicin. Chemo-and radiosensitization by ATM inhibition was not p53-dependent. Following administration to mice, KU59403 distributed to tissues and concentrations exceeding those required for in vitro activity were maintained for at least 4 hours in tumor xenografts. KU59403 significantly enhanced the antitumor activity of topoisomerase poisons in mice bearing human colon cancer xenografts (SW620 and HCT116) at doses that were nontoxic alone and well-tolerated in combination. Chemosensitization was both dose-and scheduledependent. KU59403 represents a major advance in ATM inhibitor development, being the first compound to show good tissue distribution and significant chemosensitization in in vivo models of human cancer, without major toxicity. KU59403 provides the first proof-of-principle preclinical data to support the future clinical development of ATM inhibitors. Mol Cancer Ther; 12(6); 959-67. Ó2013 AACR.