Ionizing Radiation Induced Cell Death Research Articles

P53 promotes revival stem cells in the regenerating intestine after severe radiation injury

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells.

STEM-12. TARGETING GLIOBLASTOMA WITH A BRAIN PENETRANT DRUG THAT IMPAIRS BRAIN TUMOUR STEM CELLS

Abstract Brain tumour stem cells (BTSCs) are a population of self-renewing malignant stem cells that play an important role in glioblastoma tumour hierarchy and contribute to tumour growth, therapeutic resistance, and tumour relapse. Thus, targeting of BTSCs within the bulk of tumours represents a crucial therapeutic strategy. Here, we report a potent brain penetrant drug (AJ5939) that impairs BTSCs and impedes glioblastoma progression. We show that AJ5939 inhibits the self-renewal and growth of BTSCs harbouring a diverse range of oncogenic mutations without affecting non-oncogenic neural stem cells. Global gene expression profiling revealed significant alterations in BTSC transcriptome and attenuation of the expression of a large panel of genes involved in cell cycle progression, stemness, and DNA repair mechanisms. Mechanistically, we show that AJ5939 impairs Notch signalling pathway via directly targeting this pathway and altering the stemness signature. In combination with ionizing radiation (IR), however, AJ5939 suppresses the DNA repair mechanisms and facilitate the response of the BTSC to ionizing radiation induced cell death. Finally, we present data that AJ5939 treatment of brain tumours in preclinical models significantly prolongs lifespan.

Sphingosine kinase inhibitor, SKI-II confers protection against the ionizing radiation by maintaining redox homeostasis most likely through Nrf2 signaling

Exposure to ionizing radiation (IR) set a series of deleterious events causing acute radiation syndrome and mortality, posing the need for a potent and safe radio-protective drug. IR induces cell death predominantly by causing oxidative stress and macromolecular damage. The pre-existing antioxidant defence machinery of the cellular system plays a crucial role in protecting the cells against oxidative stress by activation of Nrf2. The current study was undertaken to investigate the radio-protective potential of sphingosine kinase inhibitor (SKI-II), which was demonstrated to activate Nrf2 signaling. The safety and efficacy of SKI-II were evaluated with cell cytotoxicity, proliferation index, and clonogenic survival assays in different cell lines, namely Raw 264.7, INT-407, IEC-6 and NIH/3T3 cell lines. A safe dose of SKI-II was found radio-protective in all the cell lines linked with the activated antioxidant defence system, thereby resulting in the amelioration of IR induced oxidative stress. SKI-II pretreatment also significantly reduced DNA damage, micronuclei expression, and accelerated DNA repair kinetics as compared to IR exposed cells. Reduced oxidative stress and enhanced DNA repair significantly reduced apoptosis and suppressed the pro-death signaling associated with IR exposure. Furthermore, the in-vitro observation was verified in the in-vivo model (C57 BL/6). The Intra-peritoneal (IP) administration of SKI-II, 2 h before a lethal dose of IR exposure (7.5 Gy) resulted in 75% survival. These results imply that SKI-II ameliorates IR-induced oxidative stress and cell death by inducing anti-oxidant defence system and DNA repair pathways, thus strengthening its potential to be used as radiation countermeasure.

Effects of Radiofrequency Electromagnetic Fields and Ionizing Radiation on Amyloid Precursor Protein Processing and Cell Death

The growing concerns regarding the adverse biological effects of radiofrequency electromagnetic fields (RF-EMFs), which are generated by common electronic devices, on the human brain led us to investigate their impact on Alzheimer’s disease (AD). We aimed to establish the effects of RF-EMF on the expression of molecular markers associated with amyloid precursor protein (APP), cell death, and clonogenic survival in HT22 and APP-overexpressing 7w-PSML cells. We compared the effects of RF-EMF at a high specific absorption rate (SAR) level with the neuronal-cell-death-inducing effects of ionizing radiation (IR). RF-EMF exposure (8 W/kg SAR) promoted the protein expression of ADAM10 (α-secretase) in the HT22 cells (<i>p</i> < 0.05) and downregulated the APP mRNA level in the 7w-PSML cells (<i>p</i> < 0.01). In contrast, IR (10 Gy) significantly reduced the APP and a disintegrin and metalloproteinase 10 (ADAM10) levels without altering their respective mRNA levels in these cells. Interestingly, IR exposure significantly upregulated BACE1 (α-secretase) at both the protein and mRNA levels, suggesting adverse effects in AD. IR induced cell death and reduced clonogenic survival in both cell lines. Although RF-EMF (high SAR level) influenced APP processing, it did not induce any deleterious change in either cell line. Thus, further studies are necessary to clarify the influence of RF-EMF on AD.

Mild mitochondrial uncoupling protects from ionizing radiation induced cell death by attenuating oxidative stress and mitochondrial damage

Ionizing radiation (IR) induced mitochondrial dysfunction is associated with enhanced radiation stimulated metabolic oxidative stress that interacts randomly with intracellular bio-macromolecules causing lethal cellular injury and cell death. Since mild mitochondrial uncoupling emerged as a valuable therapeutic approach by regulating oxidative stress in most prevalent human diseases including ageing, ischemic reperfusion injury, and neurodegeneration with comparable features of IR inflicted mitochondrial damage. Therefore, we explored whether mitochondrial uncoupling could also protect from IR induced cytotoxic insult. Our results showed that DNP, BHT, FCCP, and BAM15 are safe to cells at different concentrations range depending on their respective mitochondrial uncoupling potential. Pre-incubation of murine fibroblast (NIH/3T3) cells with the safe concentration of these uncouplers followed by gamma (γ)-radiation showed significant cell growth recovery, reduced ROS generation, and apoptosis, compared to IR treatment alone. We observed that DNP pre-treatment increased the surviving fraction of IR exposed HEK-293, Raw 264.7 and NIH/3T3 cells. Additionally, DNP pre-treatment followed by IR leads to reduced total and mitochondrial oxidative stress (mos), regulated calcium (Ca2+) homeostasis, and mitochondrial bioenergetics in NIH/3T3 cells. It also significantly reduced macromolecular oxidation, correlated with the regulated ROS generation and antioxidant defence system. Moreover, DNP facilitated DNA repair kinetics evidenced by reducing the number of γ-H2AX foci formation and fragmented nuclei with time. DNP pre-incubation restrained the radiation induced pro-apoptotic factors and inhibits apoptosis. Our findings raise the possibility that mild mitochondrial uncoupling with DNP could be a potential therapeutic approach for radiation induced cytotoxic insult associated with an altered mitochondrial function.

Upregulation of NRF2 through autophagy/ERK 1/2 ameliorates ionizing radiation induced cell death of human osteosarcoma U-2 OS

The antioxidative response mediated by transcription factor NRF2 is thought to be a pivotal cellular defense system against various extrinsic stresses. It has been reported that activation of the NRF2 pathway confers cells with resistance to ionizing radiation-induced damage. However, the underlying mechanism remains largely unknown. In the current research, it was found that α-particle radiation has the ability to stimulate NRF2 expression in human osteosarcoma U-2 OS cells. Knockdown of cellular NRF2 level by shRNA-mediated gene silencing decreased the survival rate, increased the micronucleus formation rate and apoptosis rate in irradiated cells. Consistently, knockdown of NRF2 resulted in decreased expression of p65 and Bcl-2, and increased expression of p53 and Bax. Besides, it was observed that increased expression of NRF2 was partially dependent on radiation induced phosphorylation of ERK 1/2. Further results showed that radiation promoted autophagy flux which leads to the enhanced phosphorylation of ERK 1/2, as evidenced by the resultls that knockdown of ATG5 (Autophagy protein 5) gene by shRNA suppressed both radiation induced ERK 1/2 phosphorylation and NRF2 upregulation. Based on these results, it is proposed that attenuation of NRF2 antioxidative pathway can sensitize U-2 OS cells to radiation, where NRF2 antioxidative response is regulated by autophagy mediated activation of ERK 1/2 kinases.

1,4-Naphthoquinone, a pro-oxidant, ameliorated radiation induced gastro-intestinal injury through perturbation of cellular redox and activation of Nrf2 pathway.

Detrimental effects of ionizing radiation (IR) are observed at the doses above 1 Gy. Treatment modalities are available up to doses of 6 Gy including bonemarrow transplantation and administration of antibiotics. However, exposure to IR doses above 8 Gy results in gastro-intestinal (GI) syndrome characterised by denudated villi, apoptosis of crypt cells and elevated inflammatory responses. Multiple strategies have been employed to investigate novel agents to protect against IR induced injury. Since cellular redox homeostasis plays a pivotal role in deciding the cell fate, present study was undertaken to explore the potential of 1,4-naphthoquinone (NQ), a pro-oxidant, to ameliorate IR induced GI syndrome. NQ protected INT 407 cells against IR induced cell death of intestinal epithelial cells in vitro. NQ induced perturbation in cellular redox status and induced the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. Thiol antioxidant and inhibitors of Nrf2 pathway abrogated the radioprotection offered by NQ. Further, knocking down Nrf2 rescind the NQ mediated protection against IR induced cell death. In conclusion, NQ protects against IR radiation induced GI syndrome in vitro by perturbing cellular redox and activating Nrf2 pathway. This is the first report highlighting the potential of a pro-oxidant to ameliorate IR induced GI injury.

Mitochondrial induction as a potential radio-sensitizer in lung cancer cells - a short report.

Lung cancer is the leading cause of cancer death. Radiation therapy plays a key role in its treatment. Ionizing radiation induces cell death through chromosomal aberrations, which trigger mitotic catastrophe and apoptosis. However, many lung cancer patients show resistance to radiation. Dichloroacetate (DCA) is a small molecule that can promote mitochondrial activation by increasing the influx of pyruvate. Here, we tested whether DCA may increase the sensitivity of non-small cell lung cancer (NSCLC) cells to radiation through this mechanism. Two representative NSCLC cell lines (A549 and H1299) were tested for their sensitivity to radiation with and without pre-exposure to DCA. The treatment efficacy was evaluated using a clonogenic survival assay. An extracellular flux analyzer was used to assess the effect of DCA on cellular oxygen consumption as a surrogate marker for mitochondrial activity. We found that DCA increases the oxygen consumption rate in both A549 and H1299 cells by 60% (p = 0.0037) and 20% (p = 0.0039), respectively. Pre-exposure to DCA one hour before radiation increased the cytotoxic death rate 4-fold in A549 cells (55 to 13%, p = 0.004) and 2-fold in H1299 cells (35 to 17%, p = 0.28) respectively, compared to radiation alone. Mitochondrial induction by DCA may serve as a radio-sensitizer in non-small cell lung cancer.

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.

Involvement of ERK-Nrf-2 Signaling in Ionizing Radiation Induced Cell Death in Normal and Tumor Cells

Prolonged oxidative stress favors tumorigenic environment and inflammation. Oxidative stress may trigger redox adaptation mechanism(s) in tumor cells but not normal cells. This may increase levels of intracellular antioxidants and establish a new redox homeostasis. Nrf-2, a master regulator of battery of antioxidant genes is constitutively activated in many tumor cells. Here we show that, murine T cell lymphoma EL-4 cells show constitutive and inducible radioresistance via activation of Nrf-2/ERK pathway. EL-4 cells contained lower levels of ROS than their normal counterpart murine splenic lymphocytes. In response to radiation, the thiol redox circuits, GSH and thioredoxin were modified in EL-4 cells. Pharmacological inhibitors of ERK and Nrf-2 significantly enhanced radiosensitivity and reduced clonogenic potential of EL-4 cells. Unirradiated lymphoma cells showed nuclear accumulation of Nrf-2, upregulation of its dependent genes and protein levels. Interestingly, MEK inhibitor abrogated its nuclear translocation suggesting role of ERK in basal and radiation induced Nrf-2 activation in tumor cells. Double knockdown of ERK and Nrf-2 resulted in higher sensitivity to radiation induced cell death as compared to individual knockdown cells. Importantly, NF-kB which is reported to be constitutively active in many tumors was not present at basal levels in EL-4 cells and its inhibition did not influence radiosensitivity of EL-4 cells. Thus our results reveal that, tumor cells which are subjected to heightened oxidative stress employ master regulator cellular redox homeostasis Nrf-2 for prevention of radiation induced cell death. Our study reveals the molecular basis of tumor radioresistance and highlights role of Nrf-2 and ERK.

A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death.

The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of hemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids which blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its heme-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 hr before through 24 hrs after the irradiation.

IL6 Mediated Upregulation of MnSOD Protects Multiple Myeloma Cells Against Ionizing Radiation Induced Cell Death

IL6 Mediated Upregulation of MnSOD Protects Multiple Myeloma Cells Against Ionizing Radiation Induced Cell Death

Down-regulation of Wilms’ tumor 1 expression in glioblastoma cells increases radiosensitivity independently of p53

The Wilms' tumor 1 (WT1) gene is overexpressed in human glioblastoma and correlates with wild-type p53 status. In other cell types, WT1 inhibits p53-mediated apoptosis in response to DNA damaging agents. However, neither this interaction nor the relationship between WT1 and radiosensitivity has been studied in glioblastoma. To study this interaction, we generated LN-229 glioma cell lines (p53 mutant) stably expressing WT1 isoforms and induced apoptosis by transfecting with different doses of wild-type p53 plasmid expression vector. Constitutive expression of WT1 did not protect against exogenous p53-mediated apoptosis. Likewise, WT1 expression did not protect against endogenous p53-mediated cell death induced by radiotherapy in U87MG cells, which contain functional wild-type p53. We then tested the efficacy of WT1 siRNA in inhibiting WT1 expression and its effect on radiosensitivity. In T98G and LN-18 glioma cells, which possess p53 mutations, WT1 siRNA decreased WT1 protein to almost undetectable levels by 96-h post-transfection. Furthermore, WT1 siRNA transfection caused a significantly larger decrease in viability following irradiation than was seen in untransfected cells in both cell lines after treatment with ED50 of ionizing radiation. In conclusion, WT1 overexpression did not protect against p53-mediated apoptosis or ionizing radiation induced cell death. WT1 siRNA increased the radiosensitivity of two human glioma cell lines independently of p53. Anti-WT1 strategies may, therefore, prove useful in improving the response of glioblastoma to radiotherapy, thus potentially improving patient survival.

HIV Protease Inhibitors Block Akt Signaling and Radiosensitize Tumor Cells Both In vitro and In vivo

In tumor cells with mutations in epidermal growth factor receptor (SQ20B), H-Ras (T24), or K-Ras (MIAPACA2 and A549), the inhibition of Akt phosphorylation increases radiation sensitivity in clonogenic assays, suggesting that Akt is a potential molecular target when combined with therapeutic radiation. Insulin resistance and diabetes are recognized side effects of HIV protease inhibitors (HPIs), suggesting that these agents may inhibit Akt signaling. Because activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is common in human cancers, we hypothesized that HPIs can inhibit Akt activity resulting in increased tumor cell sensitivity to ionizing radiation-induced cell death. Five first-generation HPIs were subsequently tested and three of the five (amprenavir, nelfinavir, and saquinavir but not ritonavir or indinavir) inhibited Akt phosphorylation at Ser473 at serum concentrations routinely achieved in HIV patients. In both tumor cell colony formation assays and tumor regrowth delay experiments, combinations of drug and radiation exerted synergistic effects compared with either modality alone. In addition, in vivo, doses of amprenavir or nelfinavir comparable with the therapeutic levels achieved in HIV patients were sufficient to down-regulate phosphorylation of Akt in SQ20B and T24 xenografts. Finally, overexpression of active PI3K in cells without activation of Akt resulted in radiation resistance that could be inhibited with HPIs. Because there is abundant safety data on HPIs accumulated in thousands of HIV patients over the last 5 years, these agents are excellent candidates to be tested as radiation sensitizers in clinical trials.

A role of long-lived radicals in radiation mutagenesis and its suppression by epigallocatechin gallate

Ionizing radiation induces cell death, chromosome aberration and gene mutation. It is widely accepted that most of these biological effects is caused by free radicals such as hydroxyl and hydrogen radicals. However, we formerly demonstrated that ionizing radiation produces organic radicals, which have a long lifespan and whose half-life are several hours. In the present study, we studied the effect of (−)-epigallocatechin-3- O-gallate (EGCG), which scavenges the long-lived radicals on X-ray-induced gene mutation. In order to find out the role that the long-lived radicals play in radiation mutagenesis, the cells were synchronized, irradiated with 3 Gy of X-rays with a combined treatment of 200 μM EGCG for 2 h before and after DNA synthesis, and the cells mutation frequency was examined at the hypoxanthin guanine phosphoribosyl transferase ( HPRT) locus. The result indicated that the treatment with EGCG before but not after DNA synthesis post-irradiation reduced the mutation frequency. This result implies that the presence of the long-lived radicals during DNA synthesis plays a role in the induction of gene mutation by ionizing radiation.

Influence of X-ray on the autophagic-lysosomal system in rat pancreatic acini

Lysosomes have an important role in radiation injury of cells and tissues. Activation of autophagy is frequently observed in different types of pathological tissue degeneration. In radiation response it increases in some cases, and lysosomes are responsible for regulated degradation of the autophagic vacuoles. Lysosomes are also involved in ionizing radiation induced cell death. In apoptosis lysosomes degrade content of the phagocytotic vacuoles derived from engulfed apoptotic blebs. On the other hand lysosomal enzymes discharged from disintegrated cells have a key role in induction of necrotic changes. In this work we investigate autophagy and lysosomal protein degradation in the relatively radiation insensitive exocrine pancreatic acini in vivo and in vitro. Type of cell death induced by X-ray was also examined in relation to the changes of the lysosomal processes. In 5 h after 16 Gy in vivo whole body irradiation we observed significant increase in the cytoplasmic volume fraction of autophagic vacuoles and in the number of apoptotic cells in vivo. But in the acini isolated from irradiated rats we could not detect a change in the lysosomal degradation of intracellular proteins. Therefore irradiation probably influences the autophagy in an earlier step than lysosomal degradation. Extended necrotic lesions were not observed in vivo as long as 48 h. Isolated pancreatic acini usually contain more autophagic vacuoles than in vivo, but we could not observe additional increase in autophagy after 8 Gy, in vitro irradiation. Lysosomal degradation of intracellular proteins was also unaltered after 8 Gy, in vitro irradiation. Other biochemical functional parameters of the isolated pancreatic acini, like protein synthesis and amylase secretion were not changed either after 8 Gy, in vitro X-ray treatment. These results indicate that pancreatic acinar cells in vitro have a high tolerance to irradiation. The observed in vivo radiation induced changes of the exocrine pancreas are possibly indirectly induced by injuries of more sensitive mechanisms.