N-t-Butyl hydroxylamine regulates ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Therefore, compounds that scavenge reactive oxygen species may confer regulatory effects on apoptosis. Recently, it has been shown that the decomposition product of the spin-trapping agent α-phenyl-N-t-butylnitrone, N-t-butyl hydroxylamine (NtBHA), mimics α-phenyl-N-t-butylnitrone and is much more potent in delaying reactive oxygen species-associated senescence. We investigated the effects of NtBHA on ionizing radiation-induced apoptosis. Upon exposure to 2 Gy of γ-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 0.1 mM NtBHA for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function, and oxidative damage to cells. NtBHA effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The generation of intracellular reactive oxygen species was higher and the GSH level was lower in control cells compared to NtBHA-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of reactive oxygen species, and the reduction of ATP production were significantly higher in control cells compared to NtBHA-treated cells. NtBHA pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that NtBHA may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of reactive oxygen species.     

Pyridoxamine protects intestinal epithelium from ionizing radiation-induced apoptosis

Reactive oxygen species (ROS) and reactive carbonyl species (RCS) are the major causes of biological tissue damage during exposure to ionizing radiation (IR). The existing strategies to protect normal tissues from the detrimental effects of IR suffer from several shortcomings including highly toxic side effects, unfavorable administration routes, and low efficacy. These shortcomings emphasize a need for radioprotective treatments that combine effectiveness with safety and ease of use. In this paper, we demonstrate that pyridoxamine, a ROS and RCS scavenger with a very favorable safety profile, can inhibit IR-induced gastrointestinal epithelial apoptosis in cell culture and in an animal model. Pyridoxamine was more effective at protecting from radiation-induced apoptosis than amifostine, a synthetic thiol compound and the only FDA-approved radioprotector. We suggest that pyridoxamine has potential as an effective and safe radioprotective agent.     

Sensitive to apoptosis gene protein regulates ionizing radiation-induced apoptosis

Organisms exposed to ionizing radiation (IR) undergo increases in the production of reactive oxygen species (ROS), which are determinant components in the induction of apoptosis. Sensitive to apoptosis gene (SAG) encodes a redox-inducible and apoptosis-protective antioxidant protein. This report demonstrates that the modulation of SAG expression in cultured cells regulates IR-induced apoptosis. A protective role for SAG against IR-induced apoptosis was found in U937 cells transfected with SAG cDNA. A significant decrease in the endogenous production of ROS was also observed in SAG over-expressing cells, compared to control cells, exposed to 2 Gy γ-irradiation. These results suggest that SAG plays an important role in regulating IR-induced apoptosis, presumably by maintaining the cellular redox status. Because SAG is over-expressed in many human cancers, targeting SAG expression may have therapeutic value in cancer treatment. Transfection of the pancreatic cancer cell line PC3 with SAG small interfering RNA markedly attenuated the expression of SAG, augmenting their susceptibility to IR-induced apoptosis. The knockdown of SAG expression by RNA interference combined with radiotherapy may be a potential method for radiosensitization.     

Sensitization of ionizing radiation-induced apoptosis by ursolic acid

Radiation therapy has been widely used for treating human cancers. However, cancer cells develop radioresistant phenotypes that decrease the efficacy of radiotherapy. Ionizing radiation (IR) induces the production of reactive oxygen species, which play an important role in apoptotic cell death. Therefore, radiation therapy combined with a sensitizer, which modulates cellular redox status, has the potential to enhance therapeutic efficacy in a variety of human cancers. Here, we investigated the radiosensitizing effects of ursolic acid (UA), a pentacyclic triterpenoid found in rosemary and holy basil. IR-induced apoptosis in cancer cell lines such as DU145, CT26 and B16F10 was significantly enhanced by UA, as reflected by DNA fragmentation, cellular redox status, mitochondrial dysfunction and modulation of apoptotic marker proteins. Additionally, UA combined with IR was also effective for inhibiting tumorigenesis in B16F10 melanoma cells implanted into mice. Taken together, these results suggest that applying UA together with IR may be an effective combination modality for treating cancer.     

Reactive oxygen species, but not mitochondrial membrane potential, is associated with radiation-induced apoptosis of AHH-1 human lymphoblastoid cells

The aim of the study was to investigate the sensitivity of AHH-1 human lymphoblastoid cells to radiation and its relevance to intracellular events, specifically alteration in cellular energy-producing systems. AHH-1 human lymphoblastoid cells were irradiated with 6 Gy of gamma radiation, and then were collected at the indicated time points. Parallel studies were conducted to assess the effects of radiation on the cell proliferation and apoptotic index. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production were monitored. A marked decrease of cell viability was observed as early as 12 h postirradiation and fraction of apoptotic cells was highest at 24 h. Intracellular ROS generation measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) appeared to be highest as early as 30 min postirradiation and resumed to normal level at 6 h. Unexpectedly, the fluorescence intensity of Rhodamine 123 for measuring MMP did not change during the first 3h after radiation and exhibited an aberrant increase at 6 h. The results suggest that AHH-1 cells are sensitive to radiation-induced apoptosis and ROS generation is an early phase in the apoptosis process. Moreover, the results might cast doubts on those studies using Rhodamine 123 which hypothesized that the fall in MMP is one of the early events of apoptosis.     

Identification of a novel ionizing radiation-induced nuclease, AEN, and its functional characterization in apoptosis

To investigate ionizing radiation response, we screened genes that exhibit higher expression following gamma irradiation. We report here the isolation and functional characterization of a novel ionizing radiation-induced gene, AEN. Sequence analysis of AEN revealed exonuclease domain highly similar to that of exonuclease III. The AEN protein revealed DNase activity by cleaving various DNA substrates. Subcellular distribution of AEN exhibited nuclear colocalization with apoptotic nucleases such as CAD and AIF following irradiation. Moreover AEN distribution revealed perinuclear staining pattern which could be seen with other apoptotic nucleases. Irradiation of AEN-expressing cells resulted in synergistic increase of apoptosis whereas AEN deletion mutant in exonuclease domain did not. Our data, thus, suggest that radiation-induced AEN cleaves DNA in concert with other apoptotic nucleases and thereby enhances apoptosis following ionizing irradiation.     

Enhancement of radiation-induced apoptosis of human lymphoma U937 cells by sanazole

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. In this study, we determined whether sanazole enhances the radiation-induced apoptosis of human lymphoma U937 cells. Our results revealed that, compared with 10 mM sanazole or radiation alone, the combination of both resulted in a significant enhancement of apoptosis after 6 h, which was evaluated on the basis of DNA fragmentation, morphological changes, and phosphatidylserine externalization. Sanazole alone enhanced intracellular superoxide and hydrogen peroxide formation, which further increased when the cells were irradiated. Significant enhancement of Fas externalization, loss of mitochondrial membrane potential (MMP), and activation of caspase-3 and caspase-8 were observed after the combined treatment. Moreover, this combination could also enhance Bid activation, reduction of Hsp70 expression level and release of cytochrome c from the mitochondria to the cytosol. An immediate increase in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ) was observed after the combined treatment. These results suggest that the intracellular superoxide and peroxide generated by sanazole might be involved in the enhancement of radiation-induced apoptosis, and that these effects are associated with modulation of the Fas-mitochondria-caspase-dependent pathway, an increase in [Ca²⁺] _i , and a decrease in the Hsp70 expression levels.     

Simulated microgravity increases heavy ion radiation-induced apoptosis in human B lymphoblasts

Aims

Microgravity and radiation, common in space, are the main factors influencing astronauts' health in space flight, but their combined effects on immune cells are extremely limited. Therefore, the effect of simulated microgravity on heavy ion radiation-induced apoptosis, and reactive oxygen species (ROS)-sensitive apoptosis signaling were investigated in human B lymphoblast HMy2.CIR cells.

Main methods

Simulated microgravity was achieved using a Rotating Wall Vessel Bioreactor at 37 °C for 30 min. Heavy carbon-ion irradiation was carried out at 300 MeV/u, with a linear energy transfer (LET) value of 30 keV/μm and a dose rate of 1 Gy/min. Cell survival was evaluated using the Trypan blue exclusion assay. Apoptosis was indicated by Annexin V/propidium iodide staining. ROS production was assessed by cytometry with a fluorescent probe dichlorofluorescein. Malondialdehyde was detected using a kit. Extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase phosphatase-1 (MKP-1) and caspase-3 activation were measured by immunoblotting.

Key findings

Simulated microgravity decreased heavy ion radiation-induced cell survival and increased apoptosis in HMy2.CIR cells. It also amplified heavy ion radiation-elicited intracellular ROS generation, which induced ROS-sensitive ERK/MKP-1/caspase-3 activation in HMy2.CIR cells. The above phenomena could be reversed by the antioxidants N-acetyl cysteine (NAC) and quercetin.

Significance

These results illustrated that simulated microgravity increased heavy ion radiation-induced cell apoptosis, mediated by a ROS-sensitive signal pathway in human B lymphoblasts. Further, the antioxidants NAC and quercetin, especially NAC, might be good candidate drugs for protecting astronauts' and space travelers' health and safety.     

Enhancement of hyperthermia-induced apoptosis by a new synthesized class of furan-fused tetracyclic compounds

The combined effects of hyperthermia (44 degrees C, 20 min) or X-rays (10 Gy) and a new class of furan-fused tetracyclic synthesized compounds (DFs), on apoptosis in human lymphoma U937 cells were investigated. Among the tested compounds (DF1 approximately 6), the combined treatment of 10 microM DF with TIPS (triisopropylsilyloxy) (Designated #3 DF3) and hyperthermia showed the largest potency to induce DNA fragmentation at 6 h after hyperthermia but no enhancement was observed if it was combined with X-rays. Enhancement of hyperthermia-induced apoptosis by DF3 in a dose-dependent manner was observed. When the cells were treated first with DF3 at a nontoxic concentration of 20 microM, and exposed to hyperthermia afterwards, a significant enhancement of heat-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by hyperthermia, was enhanced by DF3. Mitochondrial transmembrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combination. Externalization of Fas was observed following the combined treatment. Flow cytometry revealed rapid and sustained increase of intracellular superoxide due to DF3, and showed subsequent and transient increase in the formation of intracellular hydrogen peroxide (H(2)O(2)), which was further increased when hyperthermia was combined. These results indicate that the intracellular superoxide and H(2)O(2) generated by DF3 enhance the hyperthermia-induced apoptosis via the Fas-mediated mitochondrial caspase-dependent pathway.     

Modulation of apoptosis of eckol against ionizing radiation in mice

To investigate the radioprotective potential of eckol, a component of the seaweed Ecklonia cava, against radiation in vivo, we evaluated the effect of eckol on cyto- and histo-protective capability of the lymphocytes and intestine against damage induced by a single whole body irradiation (WBI) in vivo. Here, we ascertained that eckol protected the lymphocytes’ viability and rescued intestinal cells from radiation-induced apoptosis by decreasing the amount of pro-apoptotic p53 and Bax and increasing that of anti-apoptotic Bcl-2. These findings indicate that the overexpression of anti-apoptotic protein, which may lead to resistance to DNA damage, is involved deeply in protection of gastrointestinal cells after irradiation. Thus, eckol that can protect cells and tissues against ionizing radiation may have considerable potential as adjuncts to successful radiotherapy.     

Enhancement of hyperthermia-induced apoptosis by a new synthesized class of benzocycloalkene compounds

The aim of this study was to examine whether, a new synthesized class of benzocycloalkene derivatives (BCs), enhances apoptosis induced by hyperthermia. The combined effects of hyperthermia (44°C, 20 min) and BCs on apoptosis in human lymphoma U937 cells were investigated. Among the tested compounds (BC1 ∼ 9), the combined treatment of 10 μM BC2 or BC4 and hyperthermia showed the largest potency to induce DNA fragmentation at 6 h after hyperthermia. And enhancement of hyperthermia-induced apoptosis by BC2 or BC4 in a dose-dependent manner was observed. When the cells were treated first with BC2 or BC4 at a nontoxic concentration of 20 μM, and exposed to hyperthermia afterwards, a significant enhancement of heat-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. Flow cytometry revealed an increase of intracellular superoxide due to BC2 or BC4, which was further increased when hyperthermia was combined. Mitochondrial membrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combination. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by hyperthermia, was enhanced by BC2 or BC4. An increase in the intracellular Ca²⁺ concentration [Ca²⁺]_i, externalization of Fas, and decrease in Hsp70 were observed following the combined treatment. These results indicate that the intracellular superoxide generated by BC2 or BC4 is involved in the enhancement of apoptosis through Fas-mitochondria caspase and [Ca²⁺]_i-dependent pathways, and a decrease in Hsp70 also contributed to the enhancement of apoptosis.     

The effect of α-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. α-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of γ-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP⁺+NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.     

Secretome analysis of ionizing radiation-induced senescent cancer cells reveals that secreted RKIP plays a critical role in neighboring cell migration

Cellular senescence is a physiological program of irreversible growth arrest that is considered to play an important role in tumor suppression. Recent studies demonstrated that senescent cells secrete multiple growth regulatory proteins that could alter the behavior of neighboring cells. In this study, we investigated the effect of secretory proteins from ionizing radiation (IR) induced senescent tumor cells on normal and tumor cells. Conditioned medium (CM) from IR-induced senescent MCF7 cells significantly increased cell proliferation, invasion, migration, and wound healing activity in MCF7 cells and HUVECs. Comparative proteomics analysis revealed 24 differentially secreted protein spots including Raf kinase inhibitor protein (RKIP), α-Enolase, AKAP9, and MARK4, and the findings were confirmed by Western blot analysis of IR-induced senescent cancer cells. We found that RKIP was secreted via the classical pathway, and the transfection of small interfering RNA against RKIP suppressed CM-induced migration in MCF7 cells. Treatment with recombinant human RKIP increased the migratory activity of MCF7 cells. Taken together, our results demonstrate that the senescence-associated secretory protein RKIP could be the principal target to prevent the potential effects of the secretome from IR-induced senescent tumor cells on neighboring cell migration.     

Induction of apoptosis by ionizing radiation and CI-1033 in HuCCT-1 cells

CI-1033 is a quinazoline-based HER family tyrosine kinase inhibitor that is currently being evaluated as a potential anticancer agent. The present study examines the molecular mechanism by which CI-1033 induces apoptosis either as a single agent or in combination with radiation. Although CI-1033 alone did not induce apoptosis, the simultaneous exposure of cells to CI-1033 and radiation induced significant levels of apoptosis. The sequential treatment of cells with CI-1033 followed by radiation induced an even greater effect with 62.6% of cells undergoing apoptosis but this enhanced effect was not seen if cells were treated first with radiation and then CI-1033. The combination treatment induces apoptosis of HuCCT-1 via upregulation of FasL and Bid cleavage. These data suggest that modulation of the Fas-FasL pathway and activation of Bid could be useful for increasing the anti-tumor effect of CI-1033 in this type of cancer.     

Induction of apoptosis in macrophages by cationic liposomes

The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage-like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N-acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes.     

A critical role for AKT activation in protecting cells from ionizing radiation-induced apoptosis and the regulation of acinus gene expression

Although AKT activation leads to the activation of various pathways related to cell survival, the roles of AKT in modulating cellular responses induced by ionizing radiation in normal human cells remain unclear. Here we show that low-dose radiation of 0.05 Gy did not affect cell death, but high-dose radiation (> 0.2 Gy) induced apoptosis through the activation of caspases and acinus cleavage. Ionizing radiation induced acinus phosphorylation via AKT activation. Thus, we examined the effect of AKT activation on radiation-induced cell death using CCD-18Lu cells transduced with a retroviral vector expressing constitutively active AKT (CA-AKT). The overexpression of CA-AKT rendered the cells resistant to ionizing radiation and prevented the proteolytic cleavage of acinus via phosphorylation. In addition, overexpression of CA-AKT resulted in the upregulation of acinus expression by activation of the NF-κB pathway. On the other hand, suppression of endogenous AKT expression by siRNA resulted in the reduction of acinus expression and enhanced the radiation-induced apoptosis in both CCD-18Lu and IM-9 cells. Our results suggest that AKT activation inhibits cell death during radiation-induced apoptosis through the regulation of phosphorylation and expression of acinus. The AKT/NF-κB/acinus pathway functions as one of the important regulatory mechanisms required for modulating ionizing radiation sensitivity.     

Withaferin A enhances radiation-induced apoptosis in Caki cells through induction of reactive oxygen species, Bcl-2 downregulation and Akt inhibition

Withaferin A (Wit A), a natural compound derived from the medicinal plant Withania somnifera, has been reported for the anti-tumor effects, including the inhibition of tumor cell growth, metastasis and angiogenesis. In this study, we investigated the effect of Wit A on radiation-induced apoptosis in human renal cancer cells (Caki cells). Our results showed that, compared with Wit A or radiation alone, the combination of both resulted in a significant enhancement of apoptosis, showing the increase in the cleavage of caspase-3 and PARP as well as sub-G1 cell population. In addition, reactive oxygen species (ROS) generation was correlated with the enhancement of radiation-induced apoptosis by Wit A. Wit A downregulated Bcl-2 protein levels and ectopic expression of Bcl-2 in Caki cells attenuated the apoptosis induced by Wit A plus radiation. Taken together, these results indicate that Wit A enhanced radiation-induced apoptosis in Caki cells through ROS generation, down-regulation of Bcl-2 and Akt dephosphorylation. Thus, our study shows that Wit A may be used as an effective radiosensitizer in cancer therapy.     

RALES, EPHESUS and redox

In RALES, low doses of the mineralocorticoid receptor (MR) antagonist spironolactone, added to standard of care for severe heart failure, improved survival by 30% and lowered hospitalization by 35%. Animal studies with the selective MR antagonist eplerenone have similarly shown MR blockade to prevent the cerebral, renal and coronary vascular inflammatory response to elevated aldosterone levels. There is now general acceptance that aldosterone concentrations inappropriate for salt status have major deleterious effects on the cardiovascular system.

In many instances, however (e.g. Randomized Aldactone Evaluation Study (RALES), EPHESUS) aldosterone levels are normal and salt status unremarkable and yet MR blockade has unquestioned benefits. In these instances, there is increasing evidence that coronary and cardiac MR are activated by normal circulating cortisol levels, in the cellular context of generation of reactive oxygen species (ROS) and/or alteration in intracellular redox status.

MR in VSMC and cardiomyocytes are normally predominantly occupied by cortisol in tonic inhibitory mode. Blockade of 11β hydroxysteroid dehydrogenase type II (11βHSD2) or ROS generation both serve to activate cortisol–MR complexes, thus mimicking the effects of mineralocorticoid/salt imbalance on blood vessels and the heart. In RALES and EPHESUS, it is likely that the antagonists are blocking normal levels of cortisol, not aldosterone, from activating MR in the context of tissue damage and ROS generation. If this is the case, MR antagonists may be of wide therapeutic potential in cardiovascular disease and not confined to those characterized by aldosterone/salt excess. Finally, the pathophysiologic roles of always-occupied MR in ‘unprotected’ tissues such as cardiomyocytes or neurons in response to altered intracellular redox status remain to be explored.     

Baicalin protects human skin fibroblasts from ultraviolet A radiation-induced oxidative damage and apoptosis

Reactive oxygen species (ROS) are an important factor in the development of skin photodamage after ultraviolet A (UVA) radiation. A flavonoid antioxidant, baicalin, can selectively neutralize super-oxide anion (O₂^?) while having no significant effect on ^?OH. Fibroblasts are a key component of skin dermis. In the present study, we investigated the protective effects of baicalin on human skin fibroblasts (HSFs) under UVA induced oxidative stress. Fluorescence microscopy and flow cytometry were used to assay the intracellular O₂^?, NO, ROS concentrations and the mitochondrial membrane potential. Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The concentrations of cellular MDA, SOD, GSH, T-AOC, and 8-oxo-dG were also measured. Cellular apoptosis was measured by flow cytometry and caspase-3 detection. The results revealed that UVA radiation could cause oxidative stress and apoptosis in HSFs. Interestingly, the use of baicalin after UVA radiation signi?cantly reduced the level of intracellular O₂^?, NO, and ROS, stabilized the mitochondrial membrane potential, and attenuated production of MDA and 8-oxo-dG. These ef?ciently enhanced the antioxidative defense system and protected the HSFs from subsequent oxidative stress damage and apoptosis. In other words, baicalin decreased the excessive generation of intracellular ROS and NO, and elevated the cellular antioxidative defense, which eventually mitigate the UVA-induced apoptosis. Based on our results, baicalin may have applications in the treatment of skin photodamage caused by UVA irradiation.