5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis

Radiation Countermeasures Program, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, 8901 Wisconsin Ave, Bethesda, MD 20889-5603, USA.
Journal of Radiation Research (Impact Factor: 1.8). 07/2012; 53(6). DOI: 10.1093/jrr/rrs060
Source: PubMed


The steroid androst-5-ene-3ß,17ß-diol (5-androstenediol, 5-AED) elevates circulating granulocytes and platelets in animals and humans, and enhances survival during the acute radiation syndrome (ARS) in mice and non-human primates. 5-AED promotes survival of irradiated human hematopoietic progenitors in vitro through induction of Nuclear Factor-κB (NFκB)-dependent Granulocyte Colony-Stimulating Factor (G-CSF) expression, and causes elevations of circulating G-CSF and interleukin-6 (IL-6). However, the in vivo cellular and molecular effects of 5-AED are not well understood. The aim of this study was to investigate the mechanisms of action of 5-AED administered subcutaneously (s.c.) to mice 24 h before total body γ- or X-irradiation (TBI). We used neutralizing antibodies, flow cytometric functional assays of circulating innate immune cells, analysis of expression of genes related to cell cycle progression, DNA repair and apoptosis, and assessment of DNA strand breaks with halo-comet assays. Neutralization experiments indicated endogenous G-CSF but not IL-6 was involved in survival enhancement by 5-AED. In keeping with known effects of G-CSF on the innate immune system, s.c. 5-AED stimulated phagocytosis in circulating granulocytes and oxidative burst in monocytes. 5-AED induced expression of both bax and bcl-2 in irradiated animals. Cdkn1a and ddb1, but not gadd45a expression, were upregulated by 5-AED in irradiated mice. S.c. 5-AED administration caused decreased DNA strand breaks in splenocytes from irradiated mice. Our results suggest 5-AED survival enhancement is G-CSF-dependent, and that it stimulates innate immune cell function and reduces radiation-induced DNA damage via induction of genes that modulate cell cycle progression and apoptosis.

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    • "We also have reported that radiation exposure induces elevation of circulating G-CSF and that administering a neutralizing antibody to G-CSF exacerbates the deleterious effects of radiation exposure, suggesting that G-CSF induced in response to irradiation plays an important protective role in recovery (Singh et al., 2012d). Recently, we have demonstrated that the use of the G-CSF antibody abrogates the radioprotective efficacy of few radiation countermeasures (Singh et al., 2010; Kulkarni et al., 2013; Krivokrysenko et al., 2012; Grace et al., 2012). Our current study demonstrates that DT3 is a potent stimulator of several cytokines including G-CSF, and that the radioprotective efficacy of DT3 is mediated through G-CSF. "
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    ABSTRACT: The objective of this study was to determine the cytokine induction by delta tocotrienol (DT3, a promising radiation countermeasure) and to investigate the role of granulocyte colony-stimulating factor (G-CSF) in its radioprotective efficacy against ionizing radiation in mice. Multiplex Luminex was used to analyze DT3- and other tocols (gamma-tocotrienol and tocopherol succinate)-induced cytokines in CD2F1 mice. Mice were injected with an optimal dose of DT3 and a G-CSF antibody, and their 30-day survival against cobalt-60 gamma-irradiation was monitored. Neutralization of G-CSF by administration of a G-CSF-specific antibody in DT3-injected mice was investigated by multiplex Luminex. Our data demonstrate that DT3 induced high levels of various cytokines comparable to other tocols being developed as radiation countermeasures. DT3 significantly protected mice against ionizing radiation, and administration of a G-CSF neutralizing antibody to DT3-treated animals resulted in complete abrogation of DT3's radioprotective efficacy and neutralization of G-CSF in peripheral blood. Our study findings suggest that G-CSF induced by DT3 mediates its radioprotective efficacy against ionizing radiation in mice.
    Life sciences 01/2014; 98(2). DOI:10.1016/j.lfs.2014.01.065 · 2.70 Impact Factor
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    • "Genistein administration stimulated serum granulocyte-colony stimulating factor (G-CSF) after sham irradiation or gamma-irradiation [32]. Recent study showed the radioprotective effect of some agents is mediated the through G-CSF induction [33,34]. Further previous our study also showed that G-CSF attenuated intestinal damage after radiation exposure [7]. "
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    ABSTRACT: Background Radiation therapy is the most widely used treatment for cancer, but it causes the side effect of mucositis due to intestinal damage. We examined the protective effect of genistein in tumor-bearing mice after abdominal irradiation by evaluation of apoptosis and intestinal morphological changes. Methods Mouse colon cancer CT26 cells were subcutaneously injected at the flank of BALB/c mice to generate tumors. The tumor-bearing mice were treated with abdominal radiation at 5 and 10 Gy, and with genistein at 200 mg/kg body weight per day for 1 d before radiation. The changes in intestinal histology were evaluated 12 h and 3.5 d after irradiation. To assess the effect of the combination treatment on the cancer growth, the tumor volume was determined at sacrifice before tumor overgrowth occurred. Results Genistein significantly decreased the number of apoptotic nuclei compared with that in the irradiation group 12 h after 5 Gy irradiation. Evaluation of histological changes showed that genistein ameliorated intestinal morphological changes such as decreased crypt survival, villus shortening, and increased length of the basal lamina 3.5 d after 10 Gy irradiation. Moreover, the genistein-treated group exhibited more Ki-67-positive proliferating cells in the jejunum than the irradiated control group, and crypt depths were greater in the genistein-treated group than in the irradiated control group. The mean weight of the CT26 tumors was reduced in the group treated with genistein and radiation compared with the control group. Conclusion Genistein had a protective effect on intestinal damage induced by irradiation and delayed tumor growth. These results suggest that genistein is a useful candidate for preventing radiotherapy-induced intestinal damage in cancer patients.
    BMC Complementary and Alternative Medicine 05/2013; 13(1):103. DOI:10.1186/1472-6882-13-103 · 2.02 Impact Factor
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    ABSTRACT: This study aimed to determine the role of granulocyte colony-stimulating factor (G-CSF), induced by a promising radiation countermeasure, gamma tocotrienol (GT3), in protecting mice from lethal doses of ionizing radiation. CD2F1 mice were injected with an optimal dose of GT3 and a G-CSF antibody, and their 30-d survival was monitored. An appropriate antibody isotype was used as a control. Multiplex Luminex was used to analyze GT3-induced cytokines. G-CSF neutralization by exogenous administration of a G-CSF antibody was confirmed by analyzing serum cytokine levels. Our results demonstrate that GT3 significantly protected mice against ionizing radiation, and induced high levels of G-CSF in peripheral blood 24h after administration. Injection of a G-CSF neutralizing antibody to the GT3-treated mice resulted in complete neutralization of G-CSF and abrogation of its protective efficacy. Administration of a G-CSF antibody did not affect levels of other cytokines induced by GT3. Histopathology of bone marrow from GT3-treated and -irradiated mice demonstrated protection of the hematopoietic tissue, and also that such protection was abrogated by administering a G-CSF antibody. Our results suggest that induction of high levels of G-CSF by GT3 administration is responsible for its protective efficacy against radiation injury.
    Cytokine 04/2013; 62(2). DOI:10.1016/j.cyto.2013.03.009 · 2.66 Impact Factor
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