Different Doses Of Ionizing Radiation Research Articles

Yes-Associated Protein 1 Inhibition Induces Immunogenic Cell Death and Synergizes With Radiation and PD-1 Blockade

Danger signals released by ionizing radiation (IR) can theoretically stimulate immune activation in the tumor environment (TME), but IR alone is not sufficient to induce an effective immune response in clinical practice. In this study, we investigated whether inhibition of yes-associated protein 1 (YAP1) could induce immunogenic cell death (ICD) and whether the combination of YAP1 inhibition with IR could increase in vivo immune infiltration and thereby boost a tumor response to immunotherapy. First, the expression of ICD markers, markers of T-cell activation, and key proteins involved in innate immune signaling were measured after YAP1 inhibition. Next, the expression level of YAP1 protein was measured after different doses of IR. Then, the antitumor effect of YAP1 inhibition combined with IR was investigated in vivo, and the immune status of the TME was evaluated. Finally, the efficacy of a triple therapy including YAP1 inhibition combined with IR and programmed cell death protein 1 blockade in the treatment of resistant tumors was determined. We found that YAP1 inhibition induced ICD and increased the levels of antigen presentation machinery, effectively causing the activation of T cells. Mechanistically, YAP1 inhibition induced cell DNA damage and activated the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Surprisingly, IR upregulated YAP1 expression. IR combined with YAP1 inhibition significantly inhibited cancer growth and prolonged survival, which was related to the augmented infiltration, activation, and function of CD8+ T cells in the TME. Moreover, the addition of YAP1 inhibition significantly improved the efficacy of pancreatic cancer treatment when neither radiation nor programmed cell death protein 1 inhibitors were ideal. YAP1 inhibition could trigger ICD and is a potential approach to potentiating the therapeutic efficacy of radiation therapy and anti-PD1 immunotherapy.

Urolithins increased anticancer effects of chemical drugs, ionizing radiation and hyperthermia on human esophageal carcinoma cells in vitro

Despite progress in diagnosis and treatment of esophageal cancer (EC), it is still considered as a serious malignancy with very poor prognosis. Urolithins are colonic microbiota metabolites with a wide range of pharmacological properties including chemopreventive, anti-inflammatory and anticancer activities. In this study, we hypothesized that urolithins might possess the potential to improve the efficacy of chemical drugs, ionizing radiation (IR) and/or hyperthermia on EC cells. After synthesis of urolithin A (UA), methylurolithin A (mUA) and urolithin B (UB), KYSE30 esophageal cancer cells were treated with urolithins + paclitaxel (PTX), + cisplatin (DDP), + different doses of IR or + heat-shock. Viability of cells was then determined by alamarBlue assay. To further elucidate the effects of UA, we used flow cytometry for investigation of induced apoptosis, and qRT-PCR for evaluating changes in the expression of HSP27, CCND1 and BCL2. Assessment of cell viability demonstrated that mUA increased the toxicity of PTX and DDP (up to 22.4 % and 20 %, respectively) and improved the effects of 6 Gy IR (26.5 %). Our main results achieved after UA treatment were improved toxicity of PTX and 6 Gy IR, beside enhanced effects of hyperthermia (37.3 %), which was confirmed by flow cytometry analysis and downregulation of HSP27, CCND1 and BCL2 expression. Taken together, our findings suggest that UA and mUA could be used as promising agents in combination with therapeutic modalities to improve the clinical outcomes of EC treatment.

Ripk3 Signaling Regulates Hematopoietic Stem Cell Number and Function during Stress

Background: Among all tissues, bone marrow (BM) is the most sensitive tissue to ionizing radiation (IR)-induced acute tissue damage (ATD) and chronic long-term residual damage (LT-RD). BM failure and a significant reduction in blood cells (pancytopenia) often occurs within days after exposure to IR due to the massive death of proliferative hematopoietic progenitor cells (HPCs). However, due to their quiescent cell cycle status and reduced fidelity of DNA repair feature, many hematopoietic stem cells (HSCs) cannot fully eliminate such damage and enter senescence; this results in LT-RD. Abnormal dysplastic hematopoiesis is the most common LT-RD in most victims of IR, followed by an increased risk of leukemia/lymphoma development. Thus IR exposure is an established cause of BM failure and leukemia. A significant increase in the production of inflammatory cytokines is induced by IR which contributes to the pathogenesis of both ATD and LT-RD. Such inflammatory cytokines induce the activation of Ripk3-Mlkl-mediated necroptotic signaling in HSCs. However, the role of Ripk3-Mlkl signaling in IR-induced damage has not studied. Experimental procedures: The self-renewal capacity of HSCs among Ripk3-/-, Mlkl-/- and WT mice were examined and compared by serial transplantation assay. The phenotypes of ATD and LT-RD induced by different dosages of IR were compared among Ripk3-/-, Mlkl-/- and WT mice. The mechanism by which Ripk3 signaling prevents IR-induced leukemia development was studied. Results: Ripk3-Mlkl signaling is not required for hematopoiesis during homeostatic condition. However, during serial transplantation, inactivation of such signaling prevents stress-induced loss of HSCs. Interestingly, Ripk3 signaling also induces an Mlkl-independent ROS-p38-p16-mediated senescence in HSCs. Thus Ripk3-/- HSCs showed better competitive hematopoietic ability compared to Mlkl-/- and WT HSCs during serial transplantation. A sub-lethal dosage of IR (6Gy) induces Ripk3-dependent NF-κB activation and pro-survival gene expression in HSCs, which is necessary for the survival of damaged HSCs. After 6Gy IR, although DNA damage is repaired in most HSCs within 2 days, a proportion of HSCs in WT and Mlkl-/- mice fail to fully repair the damage and undergo p53-p21-dependent senescence. However such cells in Ripk3-/- mice die from apoptosis. Thus the remaining HSCs in Ripk3-/- mice should be functionally normal, while a proportion of the remaining HSCs in Mlkl-/- and WT mice remain damaged but senescent, all as demonstrated by competitive hematopoietic reconstitution assay. Multiple low-doses of IR (1.75Gy once week × 4) induce HSC exhaustion in WT mice but not in Ripk3-/- and Mlkl-/- mice. Interestingly, almost all Ripk3-/- mice develop acute lymphoblastic leukemia within 200 days after such low dose IR, while 45% of WT and 60% of Mlkl-/- mice develop thymomas within 360 days (see Figure). Mechanistically, such low-dose IR stimulates chronic inflammatory cytokine production. Such cytokines induce Ripk3-Mlkl-mediated necroptosis in response to HSC exhaustion observed in WT mice. These cytokines also induce Ripk3-ROS-p38-p16-mediated senescence in response to impaired HSC functioning observed in both WT and Mlkl-/- mice. In Ripk3-/- mice, due to the lack of both necroptotic and senescent signaling, mutant HSCs accumulate and leukemia development is accelerated. Conclusion: Ripk3 signaling plays distinct roles in HSCs in response to different doses of IR. High-dose IR induces Ripk3-dependent NF-κB/survival signaling, which is required for the survival of HSCs which fail to repair the damage. Thus temporal inhibition of Ripk3-NF-κB signaling might help to remove the damaged HSCs thus preventing the occurrence of LT-RD. However multiple low-doses of IR induces Ripk3 activation in HSCs which represses leukemia development by inducing both ROS-p38-p16-mediated senescence and Ripk3-Mlkl-mediated necroptosis. Induced activation of Mlkl-necroptosis might help to repress leukemia development by removing damaged HSCs. Disclosures No relevant conflicts of interest to declare.

A novel superoxide dismutase mimetic sensitizes lung cancer cells to radiation and protects normal cells

Lung cancer remains the leading cause of cancer mortality worldwide, and ionizing radiation (IR) is considered one of the most successful strategies to treat such an aggressive disease. However, the large doses of IR required to effectively kill cancer cells induce significant damage to normal cells, leading to treatment failure. In the present study, we investigated the effects of GC4419, a newly developed superoxide dismutase mimetic, alone and in combination with different doses of IR on cell proliferation of normal and non‐small cell lung cancer; the molecular mechanisms of such effects were also studied. Our aim was to investigate the ability of GC4419 to prevent or minimize radiotherapy‐induced adverse effects to normal cells and to enhance the efficacy of low doses of IR to kill cancer cells. Our results revealed that treatment with GC4419 alone inhibited cancer cells proliferation without affecting the viability of normal cells. Notably, combination treatment of GC4419 and IR inhibited cell proliferation of cancer cells more significantly, when compared to IR mono therapy. DNA fragmentation and caspase cleavage were significantly increased following combined GC4419 and IR treatment, indicating activation of apoptosis pathways. Furthermore, GC4419 or IR alone increased the ratio of Bax/Bcl‐2 protein expression, indicating induction of apoptosis. Such an effect was significantly increased following the combined therapy. In conclusion, our study demonstrates that GC4419 can be used as an effective adjuvant to radiation therapy to enhance the efficacy of low dose IR and to protect normal cells against radiation‐induced damage.Support or Funding InformationNIH grants # HL 135648, HL 131941, and EB 016096 to Jay L. ZweierThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 451: Radiation Induced Atherosclerosis on Mice

Rationale: The high prevalence of cardiovascular disease (CVD) in cancer survivors after ionizing radiation (IR) therapy has long been recognized, but the mechanism for this is unknown. Previous studies reported that the plaques in IR therapy survivors exhibit characteristics of increased instability without changes in the plaque size. Objective: To study the mechanism of IR induced CVD in humans, we decided to develop an IR mouse model that mimics human CV events. Methods and Results: We fed LDLR-/- mice or C57 mice with a single injection of adeno -associated virus vector(AAV) encoding a gain-of function of PCSK9 with a high fat diet, then irradiated the mice with different doses of IR (2 Gy, 5 Gy, 10 Gy). Whole body radiation was given 2 Gy, while localized IR to the neck and thoracic area was given at 5 Gy (1 time) or 10Gy (weekly 5 Gy, twice).About 2~3 weeks after the IR treatment, we performed left partial carotid artery ligation (PCL), and 3 weeks later examined the extent of plaque formation and cardiac abnormality by comparing those detected in non-irradiated control mice. The lesion area ratio between the partially ligated left carotid artery (LCA) and the right carotid artery (RCA) was significantly increased in the IR-treated group compared to the non-IR-treated group (3.1 ± 0.3 vs.1.8 ± 0.4, n = 7-8, p = 0.03). In addition, the lesion in the IR-treated of mice had significantly larger necrotic cores with a thinner fibrous cap when compared to those in non-irradiate mice. Furthermore, IR-treated mice showed cardiac hypertrophy, in which we found a significant increase of the wall thickness of mid-to small sized arteries as well as perivascular fibrosis, which were not noticed in the non-IR-treated mice. Conclusions: Our results demonstrate that IR not only increases atherosclerotic and vulnerable plaque formation but also cardiac hypertrophy with mid- and micro-vascular wall swelling. The increase of mid- and micro-vascular wall thickness may explain the heart failure preserved heart failure, which is observed in the majority of heart failure patients after IR treatment.

NOS2 deficiency has no influence on the radiosensitivity of the hematopoietic system

ObjectivePrevious studies have shown that inhibition of inducible NO synthase (NOS2 or iNOS) with an inhibitor can selectively protect several normal tissues against radiation during radiotherapy. However, the role of NOS2 in ionizing radiation (IR)-induced bone marrow (BM) suppression is unknown and thus was investigated in the present study using NOS2−/− and wild-type mice 14 days after they were exposed to a sublethal dose of total body irradiation (TBI).MethodsThe effects of different doses of IR (1, 2 and 4 Gy) on the apoptosis and colony-forming ability of bone marrow cells from wild-type (WT) and NOS2−/− mice were investigated in vitro. In addition, we exposed NOS2−/− mice and WT mice to 6-Gy TBI or sham irradiation. They were euthanized 14 days after TBI for analysis of peripheral blood cell counts and bone marrow cellularity. Colony-forming unit-granulocyte and macrophage, burst-forming unit-erythroid and CFU-granulocyte, erythroid, macrophage in bone marrow cells from the mice were determined to evaluate the function of hematopoietic progenitor cells (HPCs), and the ability of hematopoietic stem cells (HSCs) to self-renew was analysed by the cobblestone area forming cell assay. The cell cycling of HPCs and HSCs were measured by flow cytometry.ResultsExposure to 2 and 4 Gy IR induced bone marrow cell apoptosis and inhibited the proliferation of HPCs in vitro. However, there was no difference between the cells from WT mice and NOS2−/− mice in response to IR exposure in vitro. Exposure of WT mice and NOS2−/− mice to 6 Gy TBI decreased the white blood cell, red blood cell, and platelet counts in the peripheral blood and bone marrow mononuclear cells, and reduced the colony-forming ability of HPCs (P < 0.05), damaged the clonogenic function of HSCs. However, these changes were not significantly different in WT and NOS2−/− mice.ConclusionThese data suggest that IR induces BM suppression in a NOS2-independent manner.

Low dose radiation-induced adaptive response preventing HPRT mutation is Fhit independent

Purpose: To study whether fragile histidine triad (Fhit) prevents IR-induced hypoxanthineguanine phosphoribosyltransferase (HPRT) mutation and whether Fhit plays any role in preventing HPRT mutation through low dose-induced adaptive response.Materials and methods: Establishing human cell lines with or without Fhit expression by making constructs expressing hemagglutinin (HA) alone or HA-Fhit fusion protein and transfecting the vector to HeLa cells. The effects of Fhit on ionising radiation (IR)-induced mutation were examined by observing HPRT mutation rates in the established cell lines following different doses of IR. The role of Fhit on low dose IR-induced adaptive response were examined by observing HPRT mutation rates in the established cell lines that were exposed to 0.1 Gy and followed with high dose IR or ultraviolet (UV) exposure.Results: Low dose (0.1 Gy) does not affect HPRT mutation rates in these cell lines. Fhit prevents high dose IR (≥2 Gy)-induced mutation as it prevents UV-induced mutation. However, low dose of IR (0.1 Gy)-induced adaptive response prevents both high doses of IR and UV-induced mutation in both the cells with and without Fhit expression.Conclusions: Fhit prevents IR-induced HPRT mutation and preventing mutation through low dose of IR-induced adaptive response is Fhit independent.