Further Characterization of the Mitigation of Radiation Lethality by Protective Wounding

There continues to be a major effort in the United States to develop mitigators for the treatment of mass casualties that received high-intensity acute ionizing radiation exposures from the detonation of an improvised nuclear device during a radiological terrorist attack. The ideal countermeasure should be effective when administered after exposure, and over a wide range of absorbed doses. We have previously shown that the administration of a subcutaneous incision of a defined length, if administered within minutes after irradiation, protected young adult female C57BL/6 mice against radiation-induced lethality, and increased survival after total-body exposure to an LD50/30 X-ray dose from 50% to over 90%. We refer to this approach as "protective wounding". In this article, we report on our efforts to further optimize, characterize and demonstrate the validity of the protective wounding response by comparing the response of female and male mice, varying the radiation dose, the size of the wound, and the timing of wounding with respect to administration of the radiation dose. Both male and female mice that received a subcutaneous incision after irradiation were significantly protected from radiation lethality. We observed that the extent of protection against lethality after an LD50/30 X-ray dose was independent of the size of the subcutaneous cut, and that a 3 mm subcutaneous incision is effective at enhancing the survival of mice exposed to a broad range of radiation doses (LD15-LD100). Over the range of 6.2-6.7 Gy, the increase in survival observed in mice that received an incision was associated with an enhanced recovery of hematopoiesis. The enhanced rate of recovery of hematopoiesis was preceded by an increase in the production of a select group of cytokines. Thus, a thorough knowledge of the timing of the cytokine cascade after wounding could aid in the development of novel pharmacological radiation countermeasures that can be administered several days after the actual radiation exposure

Ionizing Radiation Affects Epigenetic Programming in Young Adult Mice

Humans are exposed to low and mild doses of radiation frequently, ranging from the natural environment to medical procedures like x-ray and CT scans. Ionizing radiation of various doses has been known to cause not only cellular and genomic changes, but specific neurological systems such as the limbic system have been indicated to be particularly vulnerable. Here, we demonstrated that epigenetics is also altered by radiation. Epigenetics is a subtle chemical coding above the gene, which plays a critical role in brain development, and downstream can cause the onset of cognitive aberrations and other neurological impairments. How radiation as an external environmental factor causes epigenetic changes is not clearly understood. DNA methylation, including 5-methylcytosine (5M) and 5-hydroxymethylcytosine (5-hmC) have been shown to either suppress or activate gene transcription and as such are key epigenetic players. To elucidate the role of radiation in epigenetic outcomes, we examined epigenetic, phenotypic and transcriptional markers via immunohistochemistry, in the hippocampus and cortex. In this study C57BL/6 mouse (postnatal day 21 (P21)) began a 4-week radiation treatment of various doses totaling (2Gy-4.5Gy) via global head targeting CT exposure. We found a loss of 5M and 5-hmC as well as transcriptional markers within regions of the hippocampus and cortex. There was a significant decrease in cell proliferation in the hippocampus- specifically, in the region responsible for adult neurogenesis. The cingulate cortex (a region adjacent to the hippocampus) also exhibited dramatic alterations in several epigenetic and transcriptional markers, indicating the vulnerability of the limbic system in radiation exposure. Understanding the mechanism by which ionizing radiation affects epigenetic programming will provide insight into the transmissibility of external factors to biological systems. Additionally, this work can aid the development of protective strategies against the harmful risks associated with radiation exposure

Ionizing Radiation Affects Epigenetic Programming in Adolescent Mice

poster abstractHumans are exposed to low and mild doses of radiation frequently, ranging from the natural environment to medical procedures like x-ray and CT scans. Ionizing radiation of various doses has been known to potentially cause not only cellular but also genomic changes. Here, we demonstrate that epigenetics is also altered by the radiation. Epigenetics is a chemical coding above the gene, which plays critical roles in brain development, cognitive aberrations and other neurological impairments. How radiation, as an external environmental factor, causes epigenetic change is not understood. DNA methylation, key in epigenetics, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been shown to either suppress or activate gene transcription. To aid in elucidating the role in which radiation affects epigenetic outcomes, we examined the effects of radiation on both epigenetic and phenotypic markers within the hippocampus. In this study we treated, via x-ray C57BL/6 mice, postnatal day (P) 21 with various doses (2Gy-4.5Gy) of radiation coupled with varying frequencies (0.5 Gy x 4, 1.5 Gy x 3, or 4.5Gy x 1) during a 4-week period. We used immunohistochemistry staining with cell proliferation, transcription and epigenetic markers. We found loss of 5mC in the sub-granular layer of the dentate gyrus (DG) in the upper and lower arms. Likewise a loss of 5hmC in the sub-granular layer of the DG, as well as in the cornu Ammonis (CA) layers 1 and 2. There was also loss of a transcriptional activation marker within the DG of the hippocampus. Furthermore, decreased cell proliferation in the adult neurogenesis in the hippocampus was found. Exposure to ionizing radiation altered the normal epigenetic profile of the mice. Understanding the mechanism by which ionizing radiation affects epigenetic programming will provide insight into how to develop protection against the potentially harmful risks associated with radiation exposure

DNA damage response (DDR) pathway engagement in cisplatin radiosensitization of non-small cell lung cancer

Non-small cell lung cancers (NSCLC) are commonly treated with a platinum-based chemotherapy such as cisplatin (CDDP) in combination with ionizing radiation (IR). Although clinical trials have demonstrated that the combination of CDDP and IR appear to be synergistic in terms of therapeutic efficacy, the mechanism of synergism remains largely uncharacterized. We investigated the role of the DNA damage response (DDR) in CDDP radiosensitization using two NSCLC cell lines. Using clonogenic survival assays, we determined that the cooperative cytotoxicity of CDDP and IR treatment is sequence dependent, requiring administration of CDDP prior to IR (CDDP-IR). We identified and interrogated the unique time and agent-dependent activation of the DDR in NSCLC cells treated with cisplatin-IR combination therapy. Compared to treatment with CDDP or IR alone, CDDP-IR combination treatment led to persistence of γH2Ax foci, a marker of DNA double-strand breaks (DSB), for up to 24h after treatment. Interestingly, pharmacologic inhibition of DDR sensor kinases revealed the persistence of γ-H2Ax foci in CDDP-IR treated cells is independent of kinase activation. Taken together, our data suggest that delayed repair of DSBs in NSCLC cells treated with CDDP-IR contributes to CDDP radiosensitization and that alterations of the DDR pathways by inhibition of specific DDR kinases can augment CDDP-IR cytotoxicity by a complementary mechanism

Irradiated Human Endothelial Progenitor Cells Induce Bystander Killing in Human Non-Small Cell Lung and Pancreatic Cancer Cells

Purpose To investigate whether irradiated human endothelial progenitor cells (hEPCs) could induce bystander killing in the A549 non-small cell lung cancer (NSCLC) cells and help explain the improved radiation-induced tumor cures observed in A549 tumor xenografts co-injected with hEPCs. Materials and Methods We investigated whether co-injection of CBM3 hEPCs with A549 NSCLC cells would alter tumor xenograft growth rate or tumor cure after a single dose of 0 or 5 Gy of X-rays. We then utilized dual chamber Transwell dishes, to test whether medium from irradiated CBM3 and CBM4 hEPCs would induce bystander cell killing in A549 cells, and as an additional control, in human pancreatic cancer MIA PaCa-2 cells. The CBM3 and CBM4 hEPCs were plated into the upper Transwell chamber and the A549 or MIA PaCa-2 cells were plated in the lower Transwell chamber. The top inserts with the CBM3 or CBM4 hEPCs cells were subsequently removed, irradiated, and then placed back into the Transwell dish for 3 h to allow for diffusion of any potential bystander factors from the irradiated hEPCs in the upper chamber through the permeable membrane to the unirradiated cancer cells in the lower chamber. After the 3 h incubation, the cancer cells were re-plated for clonogenic survival. Results We found that co-injection of CBM3 hEPCs with A549 NSCLC cells significantly increased the tumor growth rate compared to A549 cells alone, but paradoxically also increased A549 tumor cure after a single dose of 5 Gy of X-rays (P < 0.05). We hypothesized that irradiated hEPCs may be inducing bystander killing in the A549 NSCLC cells in tumor xenografts, thus improving tumor cure. Bystander studies clearly showed that exposure to the medium from irradiated CBM3 and CBM4 hEPCs induced significant bystander killing and decreased the surviving fraction of A549 and MIA PaCa-2 cells to 0.46 (46%) ± 0.22 and 0.74 ± 0.07 (74%) respectively (P < 0.005, P < 0.0001). In addition, antibody depletion studies demonstrated that the bystander killing induced in both A549 and MIA PaCa-2 cells was mediated by the cytokines TNF-α and TGF-β (P < 0.05). Conclusions These data provide evidence that irradiated hEPCs can induce strong bystander killing in A549 and MIA PaCa-2 human cancer cells and that this bystander killing is mediated by the cytokines TNF-α and TGF-β

Knockdown of the DNA repair and redox signaling protein Ape1/ Ref-1 blocks ovarian cancer cell and tumor growth

Apurinic endonuclease 1/redox effector factor-1 (Ape1/Ref-1 or Ape1) is an essential protein with two distinct functions. It is a DNA repair enzyme in the base excision repair (BER) pathway and a reduction–oxidation (redox) signaling factor maintaining transcription factors in an active reduced state. Our laboratory previously demonstrated that Ape1 is overexpressed in ovarian cancer and potentially contributes to resistance. Therefore, we utilized siRNA technology to knockdown protein levels of Ape1 in ovarian cancer cell line, SKOV-3x. Knocking Ape1 down had dramatic effects on cell growth in vitro but was not due to an increase in apoptosis and at least partially due to an extension in transit time through S-phase. Similarly, human ovarian tumor xenografts with reduced levels of Ape1 protein demonstrated a dramatic reduction in tumor volume (p < 0.01) and also statistically significant (p = 0.02) differences in 18F-fluorodeoxyglucose (FDG) uptake indicating reduced glucose metabolism and cellular proliferation. Ape1's role in DNA repair and redox signaling is important to our basic understanding of ovarian cancer cell growth and these findings strongly support Ape1 as a therapeutic target

Single-Limb Irradiation Induces Local and Systemic Bone Loss in a Murine Model

Increased fracture risk is commonly reported in cancer patients receiving radiotherapy, particularly at sites within the field of treatment. The direct and systemic effects of ionizing radiation on bone at a therapeutic dose are not well-characterized in clinically relevant animal models. Using 20-week-old male C57Bl/6 mice, effects of irradiation (right hindlimb; 2 Gy) on bone volume and microarchitecture were evaluated prospectively by microcomputed tomography and histomorphometry and compared to contralateral-shielded bone (left hindlimb) and non-irradiated control bone. One week postirradiation, trabecular bone volume declined in irradiated tibias (-22%; p < 0.0001) and femurs (-14%; p = 0.0586) and microarchitectural parameters were compromised. Trabecular bone volume declined in contralateral tibias (-17%; p = 0.003), and no loss was detected at the femur. Osteoclast number, apoptotic osteocyte number, and marrow adiposity were increased in irradiated bone relative to contralateral and non-irradiated bone, whereas osteoblast number was unchanged. Despite no change in osteoblast number 1 week postirradiation, dynamic bone formation indices revealed a reduction in mineralized bone surface and a concomitant increase in unmineralized osteoid surface area in irradiated bone relative to contralateral and non-irradiated control bone. Further, dose-dependent and time-dependent calvarial culture and in vitro assays confirmed that calvarial osteoblasts and osteoblast-like MC3T3 cells were relatively radioresistant, whereas calvarial osteocyte and osteocyte-like MLO-Y4 cell apoptosis was induced as early as 48 hours postirradiation (4 Gy). In osteoclastogenesis assays, radiation exposure (8 Gy) stimulated murine macrophage RAW264.7 cell differentiation, and coculture of irradiated RAW264.7 cells with MLO-Y4 or murine bone marrow cells enhanced this effect. These studies highlight the multifaceted nature of radiation-induced bone loss by demonstrating direct and systemic effects on bone and its many cell types using clinically relevant doses; they have important implications for bone health in patients treated with radiation therapy

Responses to the 2017 ‘1 Million Gray Question’: ASTRO membership’s opinions on the most important research question facing radiation oncology

At the American Society for Radiation Oncology's (ASTRO's) 2017 annual meeting in San Diego, CA, attendees were asked, “What is the most important research question that needs to be answered in the next 3 to 5 years?” This request was meant to start a dialogue, promote thoughtful discussion within our professional community, and help inform topics for ASTRO workshops and focus meetings. Nearly 100 people responded while in attendance at the meeting, with questions that ranged from “How can we remove barriers so low- and middle-income countries can have radiation oncology facilities?” to “What is the exact role of radiation in stage IV disease in combination with immunotherapy or targeted agents to combat resistance development?” to “How can personalized care be better integrated into the oncology and radiation oncology clinical space?

DMAPT inhibits NF-κB activity and increases sensitivity of prostate cancer cells to X-rays in vitro and in tumor xenografts in vivo

Constitutive activation of the pro-survival transcription factor NF-κB has been associated with resistance to both chemotherapy and radiation therapy in many human cancers, including prostate cancer. Our lab and others have demonstrated that the natural product parthenolide can inhibit NF-κB activity and sensitize PC-3 prostate cancers cells to X-rays in vitro; however, parthenolide has poor bioavailability in vivo and therefore has little clinical utility in this regard. We show here that treatment of PC-3 and DU145 human prostate cancer cells with dimethylaminoparthenolide (DMAPT), a parthenolide derivative with increased bioavailability, inhibits constitutive and radiation-induced NF-κB binding activity and slows prostate cancer cell growth. We also show that DMAPT increases single and fractionated X-ray-induced killing of prostate cancer cells through inhibition of DNA double strand break repair and also that DMAPT-induced radiosensitization is, at least partially, dependent upon the alteration of intracellular thiol reduction-oxidation chemistry. Finally, we demonstrate that the treatment of PC-3 prostate tumor xenografts with oral DMAPT in addition to radiation therapy significantly decreases tumor growth and results in significantly smaller tumor volumes compared to xenografts treated with either DMAPT or radiation therapy alone, suggesting that DMAPT might have a potential clinical role as a radiosensitizing agent in the treatment of prostate cancer