We evaluated normal tissue specific radioprotection of the oral cavity in radiosensitive Fanconi Anemia (FA) Fancd2−/−mice with orally established tumors using mitochondrial-targeted GS-nitroxide (JP4-039). Adult (10–12 weeks old) Fancd2+/+, Fancd2+/− and Fancd2−/− mice (C57BL/6 background) and subgroups with orally established TC-1 epithelial cell tumors received a single fraction of 28 Gy or four daily fractions of 8 Gy to the head and neck. Subgroups received JP4-039 in F15 emulsion (F15/JP4-039; 0.4 mg/mouse), 4-amino-Tempo in F15 emulsion (F15/4-amino-Tempo; 0.2 mg/ mouse) or F15 emulsion alone prior to each irradiation. Oral mucosa of Fancd2−/− mice showed baseline elevated RNA transcripts for Sod2, p53, p21 and Rad51 (all P < 0.0012) and suppressed levels of Nfkb and Tgfb, (all P < 0.0020) compared with Fancd2+/+ mice. The oral mucosa in tumor-bearing mice of all genotypes showed decreased levels of p53 and elevated Tgfb and Gadd45a (P ≤ 0.0001 for all three genotypes). Intraoral F15/JP4-039, but not F15/4-amino-Tempo, modulated radiation-induced normal tissue transcript elevation, ameliorated mucosal ulceration and reduced the depletion of antioxidant stores in oral cavity tissue of all genotypes, but did not radioprotect tumors. Mitochondrial targeting makes F15/JP4-039 an effective normal tissue radioprotector for Fancd2−/− mice, as well as wild-type mice.

Background Iron overload is associated with increased severity of nonalcoholic fatty liver disease (NAFLD) including progression to nonalcoholic steatohepatitis and hepatocellular carcinoma. Aims To identify potential role(s) of iron in NAFLD, we measured its effects on pathways of oxidative stress and insulin signaling in AML-12 mouse hepatocytes. Methods Rapid iron overload was induced with 50 μM ferric ammonium citrate and 8-hydroxyquinoline. Insulin response was measured by western blot of phospho-protein kinase B. Lipid content was determined by staining with oil red O. Reactive oxygen species (ROS) were measured by flow cytometry using 5-( -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Oxidative stress was measured by western blots for phospho-jnk and phospho-p38. Results Iron increased ROS (p<0.001) and oxidative stress (p<0.001), and decreased insulin signaling by 33% (p<0.001). Treatment with stearic or oleic acids (200 μM) increased cellular lipid content and differentially modulated effects of iron. Stearic acid potentiated iron-induced ROS levels by 2-fold (p<0.05) and further decreased insulin response 59% (p<0.05) vs. iron alone. In contrast, cells treated with oleic acid were protected against iron-mediated injury; ROS levels were decreased by half (p<0.01) vs. iron alone while insulin response was restored to control (untreated) levels. The anti-oxidant curcumin reduced effects of iron on insulin signaling, ROS, and oxidative stress (p<0.01). Curcumin was similarly effective in cells treated with both stearic acid and iron. Conclusions An in-vitro model of NAFLD progression is described in which iron-induced oxidative stress inhibits insulin signaling. Pathophysiological effects of iron were increased by saturated fat and decreased by curcumin.

Hepatocellular Carcinoma (HCC) is the fifth most common cancer worldwide. β-Catenin, the central orchestrator of the canonical Wnt pathway and a known oncogene is paramount in HCC pathogenesis. Administration of phenobarbital (PB) containing water (0.05% w/v) as tumor promoter following initial injected intraperitoneal (IP) diethylnitrosamine (DEN) injection (5 µg/gm body weight) as a tumor inducer is commonly used model to study HCC in mice. Herein, nine fifteen-day male β-catenin knockout mice (KO) and fifteen wild-type littermate controls (WT) underwent DEN/PB treatment and were examined for hepatic tumorigenesis at eight months. Paradoxically, a significantly higher tumor burden was observed in KO (p<0.05). Tumors in KO were β-catenin and glutamine synthetase negative and HGF/Met, EGFR & IGFR signaling was unremarkable. A significant increase in PDGFRα and its ligand PDGF-CC leading to increased phosphotyrosine-720-PDGFRα was observed in tumor-bearing KO mice (p<0.05). Simultaneously, these livers displayed increased cell death, stellate cell activation, hepatic fibrosis and cell proliferation. Further, PDGF-CC significantly induced hepatoma cell proliferation especially following β-catenin suppression. Our studies also demonstrate that the utilized DEN/PB protocol in the WT C57BL/6 mice did not select for β-catenin gene mutations during hepatocarcinogenesis. Thus, DEN/PB enhanced HCC in mice lacking β-catenin in the liver may be due to their ineptness at regulating cell survival, leading to enhanced fibrosis and regeneration through PDGFRα activation. β-Catenin downregulation also made hepatoma cells more sensitive to receptor tyrosine kinases and thus may be exploited for therapeutics.

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Radiation oncologists have observed variation in normal tissue responses between patients in many instances with no apparent explanation. The association of clinical tissue radiosensitivity with specific genetic repair defects (Wegner’s syndrome, Ataxia telangiectasia, Bloom’s syndrome, and Fanconi anemia) has been well established, but there are unexplained differences between patients in the general population with respect to the intensity and rapidity of appearance of normal tissue toxicity including radiation dermatitis, oral cavity mucositis, esophagitis, as well as differences in response of normal tissues to standard analgesic or other palliative measures. Strategies for the use of clinical radioprotectors have included modalities designed to either prevent and/or palliate the consequences of radiosensitivity. Most prominently, modification of total dose, fraction size, or total time of treatment delivery has been necessary in many patients, but such modifications may reduce the likelihood of local control and/or radiocurability. As a model system in which to study potential radioprotection by mitochondrial-targeted antioxidant small molecules, we have studied cell lines and tissues from Fanconi anemia (Fancd2−/−) mice of two background strains (C57BL/6NHsd and FVB/N). Both were shown to be radiosensitive with respect to clonogenic survival curves of bone marrow stromal cells in culture and severity of oral cavity mucositis during single fraction or fractionated radiotherapy. Oral administration of the antioxidant GS-nitroxide, JP4-039, provided significant radioprotection, and also ameliorated distant bone marrow suppression (abscopal effect of irradiation) in Fancd2−/− mice. These data suggest that radiation protection by targeting the mitochondria may be of therapeutic benefit even in the setting of defects in the DNA repair process for irradiation-induced DNA double strand breaks.

Abstract. Background/Aim: Total-body Bone marrow transplantation is an established therapy for Fanconi anemia (FA) patients (1-4) that can result in a significant improvement in survival following donor bone marrow engraftment (4). Critical to the success of marrow engraftment has been the application of chemotherapeutic agents as a preparatory regimen for marrow transplant that minimize toxicity to the host (3).FA patients have previously been demonstrated to have a hyperactive TGF-β response pathway (5), which may be a major cause of their initial marrow failure leading to anemia, as well as their sensitivity to the regimens used to prepare for bone marrow transplantation (1-4, 6-9). FA patients are also susceptible to late post-transplant induction of leukemia and solid tumors (7-15). DNA cross-linking agents such as mitomycin-C (14), other chemotherapeutic agents and irradiation induce DNA double strand breaks and must be delivered very cautiously to FA patients. The TGF-β signaling pathway alters both baseline and post-marrow transplant hematopoiesis in FA patients and in FA animal models (16-23). The chemotherapy drug, fludarabine (6,(24)(25), has facilitated bone marrow engraftment in FA patients, who are fragile in responsiveness to agents used in preparation for marrow transplantation (1-4, 6-9). We hypothesized that agents, which ameliorate the toxicity of total-body irradiation and/or chemotherapy drugs that may reduce the induction of TGF-β (26-33) might decrease toxicity and improve engraftment in FA patient transplant recipients. Furthermore, improvement in survival of patients with FA (4) might be achieved by reduction in the toxicity of marrow transplant.In this study, FANCD2 -/-mice were used for in vitro testing of the effects of two potential modulators (JP4-039 and MMS350) of the toxicity of irradiation or each of three chemotherapeutic drugs on TGF-β induction in hematopoietic progenitor cells. FANCD2 -/-mouse marrow hematopoietic progenitors in vitro and stromal cell lines derived from the hematopoietic microenvironment (34) were used for TGF-β induction by irradiation and chemotherapy drugs. We also tested the effect of JP4-039 and MMS350 on TGF-β induction in plasma of TBI irradiated or drug-treated FANCD2 -/-(C57BL/6 background) mice. 159

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