PURPOSE: Acute side effects of radiation such as oral mucositis are observed in most patients. Although several potential radioprotective agents have been proposed, no effective agent has yet been identified. In this study, we investigated the effectiveness of synthetic compound 3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2,4-dione (KR22332) as a radioprotective agent. MATERIALS AND METHODS: Cell viability, apoptosis, the generation of reactive oxygen species (ROS), mitochondrial membrane potential changes, and changes in apoptosis-related signaling were examined in human keratinocyte (HaCaT). RESULTS: KR22332 inhibited irradiation-induced apoptosis and intracellular ROS generation, and it markedly attenuated the changes in mitochondrial membrane potential in primary human keratinocytes. Moreover, KR22332 significantly reduced the protein expression levels of ataxia telangiectasia mutated protein, p53, and tumor necrosis factor (TNF)-alpha compared to significant increases observed after radiation treatment. CONCLUSION: KR22332 significantly inhibited radiation-induced apoptosis in human keratinocytes in vitro, indicating that it might be a safe and effective treatment for the prevention of radiation-induced mucositis.
Apoptosis/drug effects/physiology ; Cell Line, Tumor ; Cell Survival/drug effects/physiology ; Humans ; Keratinocytes/metabolism ; Membrane Potential, Mitochondrial/drug effects/physiology ; Radiation-Protective Agents/chemistry/*pharmacology ; Reactive Oxygen Species/metabolism

PURPOSE: Acute side effects of radiation such as oral mucositis are observed in most patients. Although several potential radioprotective agents have been proposed, no effective agent has yet been identified. In this study, we investigated the effectiveness of synthetic compound 3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2,4-dione (KR22332) as a radioprotective agent. MATERIALS AND METHODS: Cell viability, apoptosis, the generation of reactive oxygen species (ROS), mitochondrial membrane potential changes, and changes in apoptosis-related signaling were examined in human keratinocyte (HaCaT). RESULTS: KR22332 inhibited irradiation-induced apoptosis and intracellular ROS generation, and it markedly attenuated the changes in mitochondrial membrane potential in primary human keratinocytes. Moreover, KR22332 significantly reduced the protein expression levels of ataxia telangiectasia mutated protein, p53, and tumor necrosis factor (TNF)-alpha compared to significant increases observed after radiation treatment. CONCLUSION: KR22332 significantly inhibited radiation-induced apoptosis in human keratinocytes in vitro, indicating that it might be a safe and effective treatment for the prevention of radiation-induced mucositis.
Apoptosis/drug effects/physiology ; Cell Line, Tumor ; Cell Survival/drug effects/physiology ; Humans ; Keratinocytes/metabolism ; Membrane Potential, Mitochondrial/drug effects/physiology ; Radiation-Protective Agents/chemistry/*pharmacology ; Reactive Oxygen Species/metabolism

The study examined the role of endoplasmic reticulum stress (ERS) and signaling pathways of inositol-requiring enzyme-1 (IRE1), RNA-activated protein kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) in apoptosis of mouse testicular cells treated with low-dose radiation (LDR). In the dose-dependent experiment, the mice were treated with whole-body X-ray irradiation at different doses (25, 50, 75, 100 or 200 mGy) and sacrificed 12 h later. In the time-dependent experiment, the mice were exposed to 75 mGy X-ray irradiation and killed at different time points (3, 6, 12, 18 or 24 h). Testicular cells were harvested for experiments. H(2)O(2) and NO concentrations, and Ca(2+)-ATPase activity were detected by biochemical assays, the calcium ion concentration ([Ca(2+)]i) by flow cytometry using fluo-3 probe, and GRP78 mRNA and protein expressions by quantitative real-time RT-PCR (qRT-PCR) and Western blotting, respectively. The mRNA expressions of S-XBP1, JNK, caspase-12 and CHOP were measured by qRT-PCR, and the protein expressions of IRE1α, S-XBP1, p-PERK, p-eIF2α, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP by Western blotting. The results showed that the concentrations of H2O2 and NO, the mRNA expressions of GRP78, S-XBP1, JNK, caspase-12 and CHOP, and the protein expressions of GRP78, S-XBP1, IRE1α, p-PERK, p-eIF2α, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP were significantly increased in a time- and dose-dependent manner after LDR. But the [Ca(2+)]i and Ca(2+)-ATPase activities were significantly decreased in a time- and dose-dependent manner. It was concluded that the ERS, regulated by IRE1, PERK and ATF6 pathways, is involved in the apoptosis of testicular cells in LDR mice, which is associated with ERS-apoptotic signaling molecules of JNK, caspase-12 and CHOP.
Animals ; Apoptosis ; physiology ; radiation effects ; Endoplasmic Reticulum Stress ; physiology ; radiation effects ; Male ; Mice ; Radiation ; Testis ; physiology ; radiation effects

Adopting the cell model of multilayer spherical symmetry and the circuit analysis, the present paper gives the calculated results of the voltages on each of several parts of malignant Tonsillar B-cells and Jurkat T lymphocytes when the first-order Gaussian pulses at different central frequency apposed on them. The relationship between the central frequency and the transmembrane voltages of plasma membrane is also given. The optimum frequency causing electroporation in nuclear envelope is given as well. The paper discusses the reasons of electroporation in membrane and DNA degradation in nuclear. The work provides a reference for usage of transient bipolar electric pulses in cancer treatment.
Apoptosis ; radiation effects ; B-Lymphocytes ; cytology ; radiation effects ; Cell Line, Tumor ; Cell Membrane ; physiology ; Electromagnetic Fields ; Electroporation ; methods ; Humans ; Jurkat Cells ; Nuclear Envelope ; pathology ; radiation effects

PURPOSE: The purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet (UV) light. We intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53, caspase 3 and NOXA in these processes. METHODS: Cells were irradiated with an ultraviolet lamp. Cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining and fluorescent anti-caspase 3 antibodies were used for apoptosis investigation. The quantities of p53, caspase 3, and NOXA were measured by Western blotting for to investigate the apoptosis pathway. RESULTS: Cellular toxicity on the human lens epithelium markedly increased with time after UV exposure. On Hoechst staining, we found that apoptosis also remarkably increased after exposure to ultraviolet light, compared with a control group. In the immunochemical study using anti-caspase 3 antibodies, active caspase 3 significantly increased after exposure to ultraviolet light. On Western blotting, p53 decreased, while caspase 3 and NOXA increased. CONCLUSIONS: Exposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis, which promotes the expression of NOXA and caspase 3 increases without increasing p53. This may suggest that UV induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells.
Apoptosis/*physiology ; Caspase 3/metabolism ; Cell Line ; Cell Survival/radiation effects ; Epithelial Cells/radiation effects ; Humans ; Lens, Crystalline/cytology/*physiology/*radiation effects ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Tumor Suppressor Protein p53/metabolism ; *Ultraviolet Rays

In order to investigate the effects of electric pulses on cancer cells, we carried out the experiments with exposing HepG2 and L02 to electric pulses (1 kV/cm, l00 micros, 1 Hz) for different lengths of time (8 s, 15 s, 30 s, 60 s). Annexin V-FITC Kit and Flow cytometry were used to study the apoptosis of treated cells. The results showed that the electric pulses of 1 kV/cm, l00 micros, 1 Hz for 8 s could not induce tumor cells apoptosis. Apoptosis was observed when tumor cells were stimulated for 15 s and longer, and the apoptosis percentage increased with the increase of stimulation time. Furthermore, tumor cells were more sensitive than normal cells in response to electrical pulses. Rhodamine 123 and Laser Scanning Confocal Microscope (LSCM) were used to make a real-time study of mitochondrial transmembrane potential (Deltapsim) when the tumor cells were exposed to electric pulses for 60 s. No significant change of Deltapsim was observed within 30 s stimulation. After that, the Deltapsim increased sharply and declined later, suggesting that the mitochondrial pathway may be one of the apoptosis mechanism induced by electric pulses.
Apoptosis ; radiation effects ; Electromagnetic Fields ; Hep G2 Cells ; Humans ; Membrane Potential, Mitochondrial ; physiology ; radiation effects ; Time Factors

OBJECTIVETo investigate the expressions of protein kinase C (PKC) isoforms in X-ray-exposed HepG2 cells and identify the PKC isoforms that induce radioresistance in HepG2 cells.

METHODSCultured HepG2 cells were divided into control group and 6 Gy radiation group for corresponding treatments. The fluorescence intensity (FI) and the percentage of positive cells were determined using flow cytometry.

RESULTSThe FI of PKCalpha and PKCdelta were 2.28 and 5.05 in the radiation group, respectively, significantly higher than those in the control group (P<0.05). The percentages of PKCalpha- and PKCdelta -positive cells were significantly higher in the radiation group than in the control group (P<0.05). The FI and the percentages of PKC zeta, gamma, epsilon, zeta positive cells were rather low and showed no significant differences between the two groups (P>0.05); PKCbeta expression was not detected in the two groups of cells. The apoptosis rates of the control and radiation groups were 1.73% and 20.90%, respectively.

CONCLUSIONPKCalpha and PKCdelta may be involved in protecting HepG2 cells from radiation-induced apoptosis.

Apoptosis ; physiology ; radiation effects ; Hep G2 Cells ; Humans ; Isoenzymes ; classification ; metabolism ; Protein Kinase C-alpha ; metabolism ; Protein Kinase C-delta ; metabolism ; Radiation Tolerance ; Signal Transduction ; drug effects ; physiology

Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the alpha subunit of Gs (GalphasQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GalphasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GalphasQL-stimulated Bak reporter luciferase activity. Expression of GalphasQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Galphas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Galphas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.
Apoptosis/*radiation effects ; Cell Line, Tumor ; Cyclic AMP Response Element-Binding Protein/metabolism ; GTP-Binding Protein alpha Subunits, Gs/*metabolism ; *Gamma Rays ; Heterotrimeric GTP-Binding Proteins/metabolism ; Humans ; Lung/*cytology/physiology/radiation effects ; Lung Neoplasms ; Transcription Factor AP-1/metabolism ; *Up-Regulation ; bcl-2 Homologous Antagonist-Killer Protein/*metabolism

OBJECTIVETo investigate the mechanism involved in aging process of immortalized human keratinocyte (HaCaT) and primary human epidermis keratinocyte of adults (HEKa) irradiated by ultraviolet B(UVB).

METHODSHEKa and HaCaT were repeatedly exposed to UVB at a subcytotoxic level. SA-beta-Gal staining was performed to evaluate the senescence state; flow cytometry was applied to detect the changes of apoptosis, necrosis and cell cycle. Intracellular levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by ELISA method. Western blot was performed to detect the expression pattern of redox protein p66Shc and RT-PCR was performed to determine the mRNA level of human telomerase reverse transcriptase (hTERT).

RESULTStrong positive SA-beta-Gal staining was observed in both HEKa cell and HaCaT cells after UVB irradiation. Apoptosis rate increased from (1.81 +/-0.25)% to (4.43 +/-0.28)% and necrosis rate increased from (0.05 +/-0.01)% to (0.10 +/-0.03)% in HaCaT cell, but no marked arrest of cell cycle was observed during UVB irradiation. As a contrast, apoptosis rate of in HEKa cells significantly increased from (0.65 +/-0.05)% to (59.53 +/-2.35)%, and the necrosis rate in HEKa cells also reached (3.89 +/-0.24)%(P<0.05). Growth arrest in G0/G1 phase was also found in HEKa cells. In both cell lines, intracellular level of SOD decreased and MDA increased remarkably after UVB exposure, and an increased expression of p66Shc protein was also observed. High level of hTERT mRNA was detected in HaCaT cells and UVB exposure had little effect on its expression.

CONCLUSIONThe stress-induced premature senescence (SIPS) in HaCaT and HEKa cell lines by UVB irradiation might be closely associated with increased intracellular levels of oxidative stress, not related to the telomerase expression.

Apoptosis ; Cell Line ; Cells, Immobilized ; radiation effects ; Cellular Senescence ; physiology ; radiation effects ; Humans ; Keratinocytes ; cytology ; radiation effects ; Malondialdehyde ; metabolism ; Skin ; cytology ; Superoxide Dismutase ; metabolism ; Telomerase ; genetics ; metabolism ; radiation effects ; Ultraviolet Rays ; beta-Galactosidase ; pharmacology

Exposure to light can induce photoreceptor cell death and exacerbate retinal degeneration. In this study, mice with genetic knockout of several genes, including rhodopsin kinase (Rhok-/-), arrestin (Sag-/-), transducin (Gnat1-/-), c-Fos (c-Fos-/-) and arrestin/transducin (Sag-/-/Gnat1-/-), were examined. We measured the expression levels of thousands of genes in order to investigate their roles in phototransduction signaling in light-induced retinal degeneration using DNA microarray technology and then further explored the gene network using pathway analysis tools. Several cascades of gene components were induced or inhibited as a result of corresponding gene knockout under specific light conditions. Transducin deletion blocked the apoptotic signaling induced by exposure to low light conditions, and it did not require c-Fos/AP-1. Deletion of c-Fos blocked the apoptotic signaling induced by exposure to high intensity light. In the present study, we identified many gene transcripts that are essential for the initiation of light-induced rod degeneration and proposed several important networks that are involved in pro- and anti-apoptotic signaling. We also demonstrated the different cascades of gene components that participate in apoptotic signaling under specific light conditions.
Animals ; Apoptosis/radiation effects ; G-Protein-Coupled Receptor Kinase 1/genetics ; GTP-Binding Protein alpha Subunits/genetics ; *Gene Expression Profiling ; Genes, fos/genetics ; Light/adverse effects ; Light Signal Transduction/*genetics/physiology/radiation effects ; Mice ; Mice, Knockout ; Oligonucleotide Array Sequence Analysis ; Retina/metabolism/pathology/radiation effects ; Retinal Degeneration/etiology/*genetics/physiopathology ; Transducin/genetics