Small Molecule GS-nitroxide Ameliorates the Ionizing Irradiation-induced Delay in Bone Wound Healing Measured in a Novel Murine Model

Gokhale, Abhay S.; Epperly, M.W.; Glowacki, Julie; Wang, H.; Wipf, Peter; Patrene, Kenneth D.; Dixon, Tracy; Greenberger, J.S.

doi:10.1016/j.ijrobp.2009.07.1259

Cited by 11 publications

(23 citation statements)

References 26 publications

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“…We found, however, that NTERA2 cells required an exceptionally high plating density for reproducible growth, which was not suitable for high-throughput screening. Moreover, treatment of NCCIT cells 20 min after irradiation with the previously reported radiomitigators, JP4-039 and amifostine (14) resulted in a significant reduction of caspase 3 activation (Fig. 1A), which validated the assay.…”

Section: Resultssupporting

confidence: 77%

Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

et al. 2012

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Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D0 = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D0 = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D0 = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation-induced apoptosis in NCCIT cells. Treatment of mice with a single intraperitoneal LY294002 dose of 30 mg/kg at 10 min, 4, or 24 h after LD50/30 whole-body dose of irradiation (9.25 Gy) enhanced survival. This study documents that an unbiased siRNA assay can identify new genes, signaling pathways, and chemotypes as radiation mitigators and implicate the PI3K pathway in the human radiation response.

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Section: Resultssupporting

confidence: 77%

Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

et al. 2012

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“…In addition, disruption of TGF-β signaling results in in vivo protection against ionizing radiation [57] and in increased in vitro hematopoiesis [37]. Anti-oxidant therapies were also shown to restore osteogenesis in a mouse model of impaired osseous wound healing subjected to local irradiation [58]. …”

Section: Discussionmentioning

confidence: 99%

Dysregulated in vitro hematopoiesis, radiosensitivity, proliferation, and osteoblastogenesis with marrow from SAMP6 mice

O'Sullivan

Greenberger

Goff

et al. 2012

Experimental Hematology

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The Senescence Accelerated-Prone mouse variant 6 (SAMP6) shows normal growth followed by rapid aging, development of osteopenia, and shortened lifespan, compared with control R1 mice. Because oxidative stress is a fundamental mechanism of tissue aging, we tested whether cellular parameters that are associated with oxidative stress are impaired with marrow from SAMP6 mice. We compared in vitro hematopoiesis, irradiation sensitivity, proliferative potential, and osteoblastogenesis with marrow cells from SAMP6 and R1 mice. Marrow cells from SAMP6 mice showed shortened in vitro hematopoiesis; their stromal cells showed greater radiation sensitivity and decreased proliferation. Consistent with those properties, there was constitutive upregulation of TGF-β1, an inhibitor of hematopoiesis, and of cell cycle inhibitory genes, p16INK4A and p19ARF. Paradoxically, there was constitutive expression of osteoblast genes in stromal cells from SAMP6 mice, but in vitro matrix mineralization was impaired. These studies and data included in other reports indicate that impaired proliferation of osteoblast progenitors in SAMP6 marrow may be a major factor contributing to accelerated loss of bone mass. In sum, marrow from SAMP6 mice had diminished capacity for long-term hematopoiesis, increased radiosensitivity, and reduced proliferative capacity.

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“…The drugs GS-nitroxides (JP4-039 and XJB-5-131) [1, 3], bifunctional sulfoxide (MMS-350) [11], PI3K inhibitor (LY294002) [14], and the triphenylphosphonium mitochondrial targeted imidazole fatty acid (TPP-IOA) [16] have been described. JP4-039, XJB-5-131, MMS-350 [11], BEB55 and MCF-201-89 [3] were synthesized according to published protocols and used after passing Quality Control by Liquid Chromatography/Mass Spectroscopy Standards (purity >92%) [16].…”

Section: Methodsmentioning

confidence: 99%

“…The small molecule mitochondrial targeted GS-nitroxide, JP4-039, has been shown to mitigate irradiation induced delay in bone wound healing in mice [1], protect against ionizing irradiation-induced espohagitis [2] and act as a radioprotector in mouse and human cell lines [3–4]. XJB-5-131, another mitochondrial targeted GS-nitroxide, was shown to be a radioprotector [5] and neuroprotector [10].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells

Goff

Shields

Wang

et al. 2013

Experimental Hematology

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We evaluated the use of colony formation (CFU-GM, BFU-E, and CFU-GEMM) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. Each of 11 compounds was added before (protection) or after (mitigation) ionizing irradiation including: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor (LY294002), TPP-imidazole fatty acid, (TPP-IOA), the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propanolol, and the ATP sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs, XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs that were effective in murine assays: TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide showed no significant protection or mitigation in human CB assays. These data support testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, reducing the need for animal experiments.

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Small Molecule GS-nitroxide Ameliorates the Ionizing Irradiation-induced Delay in Bone Wound Healing Measured in a Novel Murine Model

Cited by 11 publications

References 26 publications

Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

Dysregulated in vitro hematopoiesis, radiosensitivity, proliferation, and osteoblastogenesis with marrow from SAMP6 mice

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells

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