Discovering selective antiferroptotic inhibitors of the 15LOX/PEBP1 complex noninterfering with biosynthesis of lipid mediators.

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis in vitro and in vivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators in vivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.

TGFß pathway is required for viable gestation of Fanconi anemia embryos.

Overexpression of the TGFß pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFß promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFß signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFß pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFß and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFß-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFß-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFß-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.

JP4-039, a Mitochondria-Targeted Nitroxide, Mitigates the Effect of Apoptosis and Inflammatory Cell Migration in the Irradiated Mouse Retina.

We hypothesize that the injection of JP4-039, a mitochondria-targeted nitroxide, prior to irradiation of the mouse retina may decrease apoptosis and reduce neutrophil and macrophage migration into the retina. In our study, we aimed to examine the effects of JP4-039 in the mouse retina using fluorescent microscopy, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and flow cytometry. Forty-five mice and one eye per mouse were used. In Group 1, fluorescent microscopy was used to determine retinal uptake of 10 µL (0.004 mg/µL) of intravitreally injected BODIPY-labeled JP4-039 at 0, 15, and 60 min after injection. In Group 2, the TUNEL assay was performed to investigate the rate of apoptosis after irradiation in addition to JP4-039 injection, compared to controls. In Group 3, flow cytometry was used to determine the extent of inflammatory cell migration into the retina after irradiation in addition to JP4-039 injection, compared to controls. Maximal retinal uptake of JP4-039 was 15 min after intravitreal injection (p < 0.0001). JP4-039-treated eyes had lower levels of retinal apoptosis (35.8 ± 2.5%) than irradiated controls (49.0 ± 2.7%; p = 0.0066) and demonstrated reduced migration of N1 cells (30.7 ± 11.7% vs. 77.7 ± 5.3% controls; p = 0.004) and M1 cells (76.6 ± 4.2 vs. 88.1 ± 3.7% controls, p = 0.04). Pretreatment with intravitreally injected JP4-039 reduced apoptosis and inflammatory cell migration in the irradiated mouse retina, marking the first confirmed effect of this molecule in retinal tissue. Further studies may allow for safety profiling and potential use for patients with radiation retinopathy.

Redox lipid reprogramming commands susceptibility of macrophages and microglia to ferroptotic death.

Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NO•-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO• donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO• donors and/or suppression of NO• production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.

Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways.

Previous research has identified an association between external radiation and disc degeneration, but the mechanism was poorly understood. This study explores the effects of ionizing radiation (IR) on inducing cellular senescence of annulus fibrosus (AF) in cell culture and in an in vivo mouse model. Exposure of AF cell culture to 10-15 Gy IR for 5 min followed by 5 days of culture incubation resulted in almost complete senescence induction as evidenced by SA-ßgal positive staining of cells and elevated mRNA expression of the p16 and p21 senescent markers. IR-induced senescent AF cells exhibited increased matrix catabolism, including elevated matrix metalloproteinase (MMP)-1 and -3 protein expression and aggrecanolysis. Analogous results were seen with whole body IR-exposed mice, demonstrating that genotoxic stress also drives disc cellular senescence and matrix catabolism in vivo. These results have important clinical implications in the potential adverse effects of ionizing radiation on spinal health.

Intestinal Radiation Protection and Mitigation by Second-Generation Probiotic Lactobacillus-reuteri Engineered to Deliver Interleukin-22.

(1) Background: The systemic administration of therapeutic agents to the intestine including cytokines, such as Interleukin-22 (IL-22), is compromised by damage to the microvasculature 24 hrs after total body irradiation (TBI). At that time, there is significant death of intestinal microvascular endothelial cells and destruction of the lamina propria, which limits drug delivery through the circulation, thus reducing the capacity of therapeutics to stabilize the numbers of Lgr5+ intestinal crypt stem cells and their progeny, and improve survival. By its direct action on intestinal stem cells and their villus regeneration capacity, IL-22 is both an ionizing irradiation protector and mitigator. (2) Methods: To improve delivery of IL-22 to the irradiated intestine, we gavaged Lactobacillus-reuteri as a platform for the second-generation probiotic Lactobacillus-reuteri-Interleukin-22 (LR-IL-22). (3) Results: There was effective radiation mitigation by gavage of LR-IL-22 at 24 h after intestinal irradiation. Multiple biomarkers of radiation damage to the intestine, immune system and bone marrow were improved by LR-IL-22 compared to the gavage of control LR or intraperitoneal injection of IL-22 protein. (4) Conclusions: Oral administration of LR-IL-22 is an effective protector and mitigator of intestinal irradiation damage.

Combined injury: irradiation with skin or bone wounds in rodent models.

A radiation combined injury is defined as an injury that occurs in the setting of irradiation, such as those expected after a nuclear accident, radiation dispersal device release (a 'dirty bomb'), or a nuclear weapon detonation. There is much research on irradiation-associated burns and their healing, but there is less known about other injuries sustained in the context of irradiation. Animal models are limited in their correlations to clinical situations but can support research on specific questions about injuries and their healing. Mouse models of irradiation with skin or bone wounds are validated as highly reproducible and quantitative. They show dose-dependent impairment of wound healing, with later recovery. Irradiation-induced delay of bone wound healing was mitigated to different extents by single doses of gramicidin S-nitroxide JP4-039, a plasmid expressing manganese superoxide dismutase, amifostine/WR2721, or the bifunctional sulfoxide MMS-350. These models should be useful for research on mechanisms of radiation dermal and osseous damage and for further development of new radioprotectors. They also provide information of potential relevance to the effects of clinical radiation therapies.

Adipose-Derived Stem Cell Therapy Ameliorates Ionizing Irradiation Fibrosis via Hepatocyte Growth Factor-Mediated Transforming Growth Factor-ß Downregulation and Recruitment of Bone Marrow Cells.

Radiation therapy to anatomic regions, including the head and neck, chest wall, and extremities, can produce radiation-induced fibrosis (RIF). To elucidate the cellular and molecular mechanism(s) involved in RIF, female C57BL/6J mice were irradiated to the right flank to 35 Gy in single fraction using 6 Mv electrons. Radiation fibrosis was detected by day 14, was increased by day 28, and confirmed by Masson's trichrome histological staining for collagen. Biopsied tissue at day 14 showed an increase in expression of fibrosis-related genes including transforming growth factor-ß (TGF-ß) and collagens 1-6. A single adipose-derived stem cell (ASC) injection on day 28 at the irradiated site decreased by day 40: epithelial thickness, collagen deposition, and significantly improved limb excursion compared with irradiated controls. Noncontact transwell coculture of ASCs above a monolayer of irradiated human foreskin fibroblasts downregulated fibrosis-related genes TGF-ß, connective tissue growth factor, interleukin-1, NF-kB, tumor necrosis factor, and collagens 1-6. Hepatocyte growth factor (HGF) secreted by ASCs was identified as a novel mechanism by which ASCs exert antifibrotic effects by downregulating fibrotic gene expression in irradiated cells and recruiting bone marrow cells to the irradiated site. In conclusion, these data indicate a mechanistic role of HGF secreted by ASCs in reducing RIF. Stem Cells 2019;37:791-802.

Necrostatin-1 rescues mice from lethal irradiation.

There is an emerging need in new medical products that can mitigate and/or treat the short- and long-term consequences of radiation exposure after a radiological or nuclear terroristic event. The direct effects of ionizing radiation are realized primarily via apoptotic death pathways in rapidly proliferating cells within the initial 1-2days after the exposure. However later in the course of the radiation disease necrotic cell death may ensue via direct and indirect pathways from increased generation of pro-inflammatory cytokines. Here we evaluated radiomitigative potential of necrostatin-1 after total body irradiation (TBI) and the contribution of necroptosis to cell death induced by radiation. Circulating TNFα levels were increased starting on d1 after TBI and associated with increased plasmalemma permeability in ileum of irradiated mice. Necrostatin-1 given iv. 48h after 9.5Gy TBI attenuated radiation-induced receptor interacting protein kinase 3 (RIPK3) serine phosphorylation in ileum and improved survival vs. vehicle. Utilizing apoptosis resistant cytochrome c(-/-) cells, we showed that radiation can induce necroptosis, which is attenuated by RNAi knock down of RIPK1 and RIPK3 or by treatment with necrostatin-1 or -1s whereas 1-methyl-L-tryptophan, an indoleamine-2,3-dioxygenase inhibitor, did not exhibit radiomitigative effect. This suggests that the beneficial effect of necrostatin-1 is likely through inhibition of RIPK1-mediated necroptotic pathway. Overall, our data indicate that necroptosis, a form of programmed necrosis, may play a significant role in cell death contributing to radiation disease and mortality. This study provides a proof of principle that necrostatin-1 and perhaps other RIPK1 inhibitors are promising therapeutic agents for radiomitigation after TBI.

FANCD2 protects against bone marrow injury from ferroptosis.

Bone marrow injury remains a serious concern in traditional cancer treatment. Ferroptosis is an iron- and oxidative-dependent form of regulated cell death that has become part of an emerging strategy for chemotherapy. However, the key regulator of ferroptosis in bone marrow injury remains unknown. Here, we show that Fanconi anemia complementation group D2 (FANCD2), a nuclear protein involved in DNA damage repair, protects against ferroptosis-mediated injury in bone marrow stromal cells (BMSCs). The classical ferroptosis inducer erastin remarkably increased the levels of monoubiquitinated FANCD2, which in turn limited DNA damage in BMSCs. FANCD2-deficient BMSCs were more sensitive to erastin-induced ferroptosis (but not autophagy) than FANCD2 wild-type cells. Knockout of FANCD2 increased ferroptosis-associated biochemical events (e.g., ferrous iron accumulation, glutathione depletion, and malondialdehyde production). Mechanically, FANCD2 regulated genes and/or expression of proteins involved in iron metabolism (e.g., FTH1, TF, TFRC, HAMP, HSPB1, SLC40A1, and STEAP3) and lipid peroxidation (e.g., GPX4). Collectively, these findings indicate that FANCD2 plays a novel role in the negative regulation of ferroptosis. FANCD2 could represent an amenable target for the development of novel anticancer therapies aiming to reduce the side effects of ferroptosis inducers.

ATR kinase activation in G1 phase facilitates the repair of ionizing radiation-induced DNA damage.

The kinase ATR is activated by RPA-coated single-stranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells.

SARS-CoV-2 Spike Protein Induces Oxidative Stress and Senescence in Mouse and Human Lung.

BACKGROUND/AIM: There is concern that people who had COVID-19 will develop pulmonary fibrosis. Using mouse models, we compared pulmonary inflammation following injection of the spike protein of SARS-CoV-2 (COVID-19) to radiation-induced inflammation to demonstrate similarities between the two models. SARS-CoV-2 (COVID-19) induces inflammatory cytokines and stress responses, which are also common to ionizing irradiation-induced acute pulmonary damage. Cellular senescence, which is a late effect following exposure to SARS-CoV-2 as well as radiation, was investigated. MATERIALS AND METHODS: We evaluated the effect of SARS-CoV-2 spike protein compared to ionizing irradiation in K18-hACE2 mouse lung, human lung cell lines, and in freshly explanted human lung. We measured reactive oxygen species, DNA double-strand breaks, stimulation of transforming growth factor-beta pathways, and cellular senescence following exposure to SARS-CoV-2 spike protein, irradiation or SARS-COV-2 and irradiation. We also measured the effects of the antioxidant radiation mitigator MMS350 following irradiation or exposure to SARS-CoV-2. RESULTS: SARS-CoV-2 spike protein induced reactive oxygen species, DNA double-strand breaks, transforming growth factor-ß signaling pathways, and senescence, which were exacerbated by prior or subsequent ionizing irradiation. The water-soluble radiation countermeasure, MMS350, reduced spike protein-induced changes. CONCLUSION: In both the SARS-Co-2 and the irradiation mouse models, similar responses were seen indicating that irradiation or exposure to SARS-CoV-2 virus may lead to similar lung diseases such as pulmonary fibrosis. Combination of irradiation and SARS-CoV-2 may result in a more severe case of pulmonary fibrosis. Cellular senescence may explain some of the late effects of exposure to SARS-CoV-2 spike protein and to ionizing irradiation.

Metformin and adipose-derived stem cell combination therapy alleviates radiation-induced skin fibrosis in mice.

BACKGROUND: Radiation therapy often leads to late radiation-induced skin fibrosis (RISF), causing movement impairment and discomfort. We conducted a comprehensive study to assess the effectiveness of metformin and adipose-derived stem cells (ASCs), whether autologous or allogeneic, individually or in combination therapy, in mitigating RISF. METHODS: Using a female C57BL/6J mouse model subjected to hind limb irradiation as a representative RISF model, we evaluated metformin, ASCs, or their combination in two contexts: prophylactic (started on day 1 post-irradiation) and therapeutic (initiated on day 14 post-irradiation, coinciding with fibrosis symptoms). We measured limb movement, examined skin histology, and analyzed gene expression to assess treatment efficacy. RESULTS: Prophylactic metformin and ASCs, whether autologous or allogeneic, effectively prevented late fibrosis, with metformin showing promising results. However, combination therapy did not provide additional benefits when used prophylactically. Autologous ASCs, alone or with metformin, proved most effective against late-stage RISF. Prophylactic intervention outperformed late therapy for mitigating radiation skin damage. Co-culture studies revealed that ASCs and metformin downregulated inflammation and fibrotic gene expression in both mouse and human fibroblasts. CONCLUSIONS: Our study suggests metformin's potential as a prophylactic measure to prevent RISF, and the combination of ASCs and metformin holds promise for late-stage RISF treatment. These findings have clinical implications for improving the quality of life for those affected by radiation-induced skin fibrosis.

Genetically Engineered Probiotic Limosilactobacillus reuteri Releasing IL-22 (LR-IL-22) Modifies the Tumor Microenvironment, Enabling Irradiation in Ovarian Cancer.

Despite recent advances in cancer therapy, ovarian cancer remains the most lethal gynecological cancer worldwide, making it crucial and of the utmost importance to establish novel therapeutic strategies. Adjuvant radiotherapy has been assessed historically, but its use was limited by intestinal toxicity. We recently established the role of Limosilactobacillus reuteri in releasing IL-22 (LR-IL-22) as an effective radiation mitigator, and we have now assessed its effect in an ovarian cancer mouse model. We hypothesized that an LR-IL-22 gavage would enable intestinal radioprotection by modifying the tumor microenvironment and, subsequently, improving overall survival in female C57BL/6MUC-1 mice with widespread abdominal syngeneic 2F8cis ovarian cancer. Herein, we report that the LR-IL-22 gavage not only improved overall survival in mice when combined with a PD-L1 inhibitor by inducing differential gene expression in irradiated stem cells but also induced PD-L1 protein expression in ovarian cancer cells and mobilized CD8+ T cells in whole abdomen irradiated mice. The addition of LR-IL-22 to a combined treatment modality with fractionated whole abdomen radiation (WAI) and systemic chemotherapy and immunotherapy regimens can facilitate a safe and effective protocol to reduce tumor burden, increase survival, and improve the quality of life of a locally advanced ovarian cancer patient.

Mitigation of Fetal Irradiation Injury from Mid-Gestation Total Body Radiation with Mitochondrial-Targeted GS-Nitroxide JP4-039.

Victims of a radiation terrorist event will include pregnant women and unborn fetuses. Mitochondrial dysfunction and oxidative stress are key pathogenic factors of fetal irradiation injury. The goal of this preclinical study is to investigate the efficacy of mitigating fetal irradiation injury by maternal administration of the mitochondrial-targeted gramicidin S (GS)- nitroxide radiation mitigator, JP4-039. Pregnant female C57BL/6NTac mice received 3 Gy total body ionizing irradiation (TBI) at mid-gestation embryonic day 13.5 (E13.5). Using novel time- and-motion-resolved 4D in utero magnetic resonance imaging (4D-uMRI), we found TBI caused extensive injury to the fetal brain that included cerebral hemorrhage, loss of cerebral tissue, and hydrocephalus with excessive accumulation of cerebrospinal fluid (CSF). Histopathology of the fetal mouse brain showed broken cerebral vessels and elevated apoptosis. Further use of novel 4D Oxy-wavelet MRI capable of probing in vivo mitochondrial function in intact brain revealed significant reduction of mitochondrial function in the fetal brain after 3Gy TBI. This was validated by ex vivo Oroboros mitochondrial respirometry. Maternal administration JP4-039 one day after TBI (E14.5), which can pass through the placental barrier, significantly reduced fetal brain radiation injury and improved fetal brain mitochondrial respiration. This also preserved cerebral brain tissue integrity and reduced cerebral hemorrhage and cell death. As JP4-039 administration did not change litter sizes or fetus viability, together these findings indicate JP4-039 can be deployed as a safe and effective mitigator of fetal radiation injury from mid-gestational in utero ionizing radiation exposure. One Sentence Summary: Mitochondrial-targeted gramicidin S (GS)-nitroxide JP4-039 is safe and effective radiation mitigator for mid-gestational fetal irradiation injury.

Oxidized phospholipids as biomarkers of tissue and cell damage with a focus on cardiolipin.

Oxidized phospholipid species are important, biologically relevant, lipid signaling molecules that usually exist in low abundance in biological tissues. Along with their inherent stability issues, these oxidized lipids present themselves as a challenge in their detection and identification. Often times, oxidized lipid species can co-chromatograph with non-oxidized species making the detection of the former extremely difficult, even with the use of mass spectrometry. In this study, a normal-phase and reverse-phase two dimensional high performance liquid chromatography (HPLC)-mass spectrometric system was applied to separate oxidized phospholipids from their non-oxidized counterparts, allowing unambiguous detection in a total lipid extract. We have utilized bovine heart cardiolipin as well as commercially available tetralinoleoyl cardiolipin oxidized with cytochrome c (cyt c) and hydrogen peroxide as well as with lipoxygenase to test the separation power of the system. Our findings indicate that oxidized species of not only cardiolipin, but other phospholipid species, can be effectively separated from their non-oxidized counterparts in this two dimensional system. We utilized three types of biological tissues and oxidative insults, namely rotenone treatment of lymphocytes to induce mitochondrial damage and cell death, pulmonary inhalation exposure to single walled carbon nanotubes, as well as total body irradiation, in order to identify cardiolipin oxidation products, critical to the cell damage/cell death pathways in these tissues following cellular stress/injury. Our results indicate that selective cardiolipin (CL) oxidation is a result of a non-random free radical process. In addition, we assessed the ability of the system to identify CL oxidation products in the brain, a tissue known for its extreme complexity and diversity of CL species. The ability of the two dimensional HPLC-mass spectrometric system to detect and characterize oxidized lipid products will allow new studies to be formulated to probe the answers to biologically important questions with regard to oxidative lipidomics and cellular insult. This article is part of a Special Issue entitled: Oxidized phospholipids - their properties and interactions with proteins.

Pharmacologic profiling of phosphoinositide 3-kinase inhibitors as mitigators of ionizing radiation-induced cell death.

Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after IR abrogated cell death.

Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria.

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.

Chemical Carcinogen (Dimethyl-benzanthracene) Induced Transplantable Cancer in Fanconi Anemia (Fanca-/-) Mice.

BACKGROUND/AIM: Patients with radiation sensitive Fanconi anemia (FA) are presenting with cancers of the oral cavity, oropharynx, and other anatomic locations. MATERIALS AND METHODS: Animal models for cancer in FA mice used orthotopic tumors from wild type mice. We derived a cancer cell line from Fanca-/- mice by topical application of the chemical carcinogen dimethyl benzanthracene (DMBA). RESULTS: A Fanca-/- mouse rhabdomyosarcoma was derived from a Fanca-/- (129/Sv) mouse. The in vitro clonogenic survival of the Fanca-/- clone 6 cancer cell line was consistent with the FA genotype. Transplanted tumors demonstrated hypoxic centers surrounded by senescent cells. CONCLUSION: This Fanca-/- mouse syngeneic cancer should provide a valuable resource for discovery and development of new normal tissue radioprotectors for patients with FA and cancer.

Carcinogen 4-Nitroquinoline Oxide (4-NQO) Induces Oncostatin-M (OSM) in Esophageal Cells.

BACKGROUND/AIM: The earliest cellular and molecular biologic changes in the esophagus that lead to esophageal cancer were evaluated in a mouse model. We correlated numbers of senescent cells with the levels of expression of potentially carcinogenic genes in sorted side population (SP) cells containing esophageal stem cells and non-stem cells in the non-side population cells in the 4-nitroquinolone oxide (NQO)-treated esophagus. MATERIALS AND METHODS: We compared stem cells with non-stem cells from the esophagus of mice treated with the chemical carcinogen 4-NQO (100 µg/ml) in drinking water. We also compared gene expression in human esophagus samples treated with 4-NQO (100 µg/ml media) to non-treated samples. We separated and quantitated the relative levels of expression of RNA using RNAseq analysis. We identified senescent cells by luciferase imaging of p16+/LUC mice and senescent cells in excised esophagus from tdTOMp16+ mice. RESULTS: A significant increase in the levels of RNA for oncostatin-M was found in senescent cells of the esophagus from 4-NQO-treated mice and human esophagus in vitro. CONCLUSION: Induction of OSM in chemically-induced esophageal cancer in mice correlates with the appearance of senescent cells.