BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it has a poor prognosis and few therapeutic options. Radiotherapy is one of the most effective forms of cancer treatment, and P53 protein is one of the key molecules determining how a cell responds to radiotherapy. The aim of this study was to determine the therapeutic efficacy of iodine-131 in three human HCC cell lines. METHODS: Western blotting was used to measure P53 expression. The effects of radiotherapy with iodine-131 were assessed by using the clonogenic assay to evaluate cell survival. Flow cytometry was carried out to examine the effects of iodine-131 on cell death, oxidative stress, reduced intracellular glutathione expression, the mitochondrial membrane potential, and the cell cycle. RESULTS: The P53 protein was not expressed in Hep3B2.1-7 cells, was expressed at normal levels in HepG2 cells, and was overexpressed in HuH7 cells. P53 expression in the HuH7 and HepG2 cell lines increased after internal and external irradiation with iodine-131. Irradiation induced a decrease in cell survival and led to a decrease in cell viability in all of the cell lines studied, accompanied by cell death via late apoptosis/necrosis and necrosis. Irradiation with 131-iodine induced mostly cell-cycle arrest in the G0/G1 phase. CONCLUSIONS: These results suggest that P53 plays a key role in the radiotherapy response of HCC.
Apoptosis/*radiation effects ; Blotting, Western ; Carcinoma, Hepatocellular/metabolism/pathology/radiotherapy ; Cell Line, Tumor ; Cell Survival/drug effects ; G1 Phase Cell Cycle Checkpoints/radiation effects ; *Gamma Rays ; Glutathione/metabolism ; Hep G2 Cells ; Humans ; Iodine Radioisotopes/chemistry/pharmacology/therapeutic use ; Liver Neoplasms/metabolism/pathology/radiotherapy ; Phosphorylation ; Reactive Oxygen Species/metabolism ; Tumor Suppressor Protein p53/*metabolism

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncology. The hypoxia-inducible factor (HIF)-1α plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by irradiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving fraction. EGFP-LC3 plasmid was stably transfected into cells to monitor autophagic processes. Western blotting was used to evaluate the protein expression levels of HIF-1α, c-Jun, phosphorylated c-Jun, Beclin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phosphorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Beclin 1 expression, instead of HIF-1α, could be blocked by SP600125 (a specific inhibitor of c-Jun NH2-terminal kinase), followed by suppression of autophagy. Under hypoxia, combined treatment of irradiation and chloroquine (a potent autophagy inhibitor) significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclin1 expression may be considered as a target to reverse the radioresistance in cancer cells.
Animals ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; Beclin-1 ; Cell Hypoxia ; Cell Line, Tumor ; Cell Survival ; genetics ; radiation effects ; Gene Expression Regulation, Neoplastic ; radiation effects ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Immunoblotting ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Membrane Proteins ; genetics ; metabolism ; Mice, Nude ; Microscopy, Fluorescence ; Microtubule-Associated Proteins ; genetics ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-jun ; metabolism ; Radiation Tolerance ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Heterologous ; Tumor Burden ; genetics

This study is to investigate the effect and mechanism of puerarin on DNA damage of HaCaT cells induced by UVB. Puerarin pre-treated cells were irradiated with UVB at 30 mJ x cm(-2). Twenty four hours after irradiation, DNA damage was detected by comet assay, ceramide was measured by thin layer chromatography and gas chromatography, intracellular free calcium ion was analyzed by flow cytometry, the phosphorylation level of p38 protein was examined by Western blotting method. Levels of DNA damage, ceramide, free calcium ion and p-p38 protein were elevated in UVB model cells. Contrary to the model group, all indicators above were reduced in all groups pre-treated by puerarin. Puerarin restrains the ceramide accumulation to block downstream p38 MAPK pathway and calcium ion rising, therefore reduces DNA damage in HaCaT cells induced by UVB.
Calcium ; metabolism ; Cell Line ; Ceramides ; metabolism ; DNA Damage ; drug effects ; radiation effects ; Down-Regulation ; Humans ; Isoflavones ; pharmacology ; Keratinocytes ; cytology ; metabolism ; Phosphorylation ; Signal Transduction ; drug effects ; Ultraviolet Rays ; adverse effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo explore the relationship between microglial proinflammatory and electromagnetic radiation and unveil the role of microglia in microwave radiation induced central nervous system injury.

METHODSN9 microglia cells cultured in vitro were exposed to microwave at 90 mW/cm2. Cell flow cytometry was used to observe the expression of CD11b at different time points after exposure; ELISA was used to detect the concentration of TNF-alpha in N9 cell culture supernatant; RT-PCR analysis confirmed iNOS mRNA expression in N9 microglia cells; and Nitrate Reductase Method was used to test NO amount in culture supernatant.

RESULTSThe CD11b positive microglial cells increased significantly at 3 h after microwave exposure (P < 0.05), continued to increase until 24 h and peaked at 6 h after exposure. The amount of TNF-alpha rose dramatically from 1 h to 24 h after exposure (P < 0.01) and peaked at 3 h [(762.1 +/- 61.5) pg/ml] after exposure (P < 0.01). The level of NO started to increase at 1 h [(4.48-0.59) micromol/L] and lasted for 24 h after exposure. The expression of iNOS mRNA increased significantly at 1 h (P < 0.05), and tripled the original expression at 6 h after exposure, hereafter, it decreased slightly, but all were higher than the control group within 24 h after exposure.

CONCLUSIONMicrowave radiation could induce the activation of microglia cells. The activated microglia cells could induce microglial proinflammatory by producing large amounts of TNF-alpha, NO, etc.

Animals ; Cell Line ; Cells, Cultured ; Mice ; Microglia ; metabolism ; radiation effects ; Microwaves ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Phosphorylation ; RNA, Messenger ; genetics ; Tumor Necrosis Factors ; metabolism

Skin exposure to low-dose ultraviolet B (UVB) light up-regulates the expression of matrix metalloproteinase-1 (MMP-1), thus contributing to premature skin aging (photo-aging). Although cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2), have been associated with UVB-induced signaling to MMP expression, very little are known about the roles of lipoxygenases and their products, especially leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), in MMP-1 expression in skin keratinocytes. In the present study, we demonstrate that BLT2, a cell surface receptor for LTB4 and 12(S)-HETE, plays a critical role in UVB-mediated MMP-1 upregulation in human HaCaT keratinocytes. Moreover, our results demonstrated that BLT2-mediated MMP-1 upregulation occurs through a signaling pathway dependent on reactive oxygen species (ROS) production and the subsequent stimulation of ERK. Blockage of BLT2 via siRNA knockdown or with the BLT2-antagonist LY255283 completely abolished the up-regulated expression of MMP-1 induced by low-dose UVB irradiation. Finally, when HaCaT cells were transiently transfected with a BLT2 expression plasmid, MMP-1 expression was significantly enhanced, along with ERK phosphorylation, suggesting that BLT2 overexpression alone is sufficient for MMP-1 up-regulation. Together, our results suggest that the BLT2-ROS-ERK-linked cascade is a novel signaling mechanism for MMP-1 upregulation in low-dose UVB-irradiated keratinocytes and thus potentially contributes to photo-aging.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/biosynthesis ; Cell Line ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Keratinocytes/metabolism/*radiation effects ; Leukotriene B4/biosynthesis ; Matrix Metalloproteinase 1/*biosynthesis ; Phosphorylation ; Reactive Oxygen Species/metabolism ; Receptors, Leukotriene B4/*physiology ; Signal Transduction ; Ultraviolet Rays/*adverse effects

In eukaryotic cells, DNA damage triggers activation of checkpoint signaling pathways that coordinate cell cycle arrest and repair of damaged DNA. These DNA damage responses serve to maintain genome stability and prevent accumulation of genetic mutations and development of cancer. The p38 MAPK was previously implicated in cellular responses to several types of DNA damage. However, the role of each of the four p38 isoforms and the mechanism for their involvement in DNA damage responses remained poorly understood. In this study, we demonstrate that p38γ, but not the other p38 isoforms, contributes to the survival of UV-treated cells. Deletion of p38γ sensitizes cells to UV exposure, accompanied by prolonged S phase cell cycle arrest and increased rate of apoptosis. Further investigation reveal that p38γ is essential for the optimal activation of the checkpoint signaling caused by UV, and for the efficient repair of UV-induced DNA damage. These findings have established a novel role of p38γ in UV-induced DNA damage responses, and suggested that p38γ contributes to the ability of cells to cope with UV exposure by regulating the checkpoint signaling pathways and the repair of damaged DNA.
Animals ; Apoptosis ; Cell Cycle Proteins ; metabolism ; Cells, Cultured ; DNA Damage ; DNA Repair ; Enzyme Activation ; Fibroblasts ; metabolism ; radiation effects ; Gene Deletion ; Histones ; metabolism ; Mice ; Mitogen-Activated Protein Kinase 12 ; genetics ; metabolism ; Phosphorylation ; S Phase ; Tumor Suppressor Protein p53 ; metabolism ; Ultraviolet Rays

AIMTo study the development of changes for signaling molecules related to Raf/MEK/ERK pathway in hippocampus of rats after electromagnetic radiation, and investigate the mechanisms of radiation injury.

METHODSRats were exposed to X-HPM, S-HPM and EMP radiation source respectively, and animal model of electromagnetic radiation was established. Western blot was used to detect the expression of Raf-1, phosphorylated Raf-1 and phospholylated ERK.

RESULTSThe expression of Raf-1 down-regulated during 6 h-14 d after radiation, most significantly at 7 d, and recovered at 28 d. There was no significant difference between the radiation groups. The expression of phosphorylated Raf-1 and phosphorylated ERK both up-regulated at 6 h and 7 d after radiation, more significantly at 6 h, and the two microwave groups were more serious for phosphorylated ERK. During 6 h-14 d after S-HPM radiation, the expression of phosphorylated Raf-1 increased continuously, but phosphorylated ERK changed wavily, 6 h and 7 d were expression peak.

CONCLUSIONRaf/MEK/ERK signaling pathway participates in the hippocampus injury induced by electromagnetic radiation. The excessive activation of ERK pathway may result in the apoptosis and death of neurons, which is the important mechanism of recognition disfunction caused by electromagnetic radiation.

Animals ; Apoptosis ; Electromagnetic Radiation ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hippocampus ; metabolism ; physiopathology ; radiation effects ; MAP Kinase Kinase Kinases ; metabolism ; MAP Kinase Signaling System ; radiation effects ; Male ; Phosphorylation ; Proto-Oncogene Proteins c-raf ; metabolism ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVETo observe the effect of mitogen activated protein kinase (MAPK) signal transduction system on the apoptosis induced by electromagnetic exposure in PC12 cells.

METHODSAfter pretreated by SB203580 alone or together with U0126, PC12 cells were exposed to 65 mW/cm(2) electromagnetic wave for 20 min. The phosphorylations of ERK1/2, JNK and P38 MAPK were tested by Western-blot at 3 h and 24 h after electromagnetic exposure. The apoptosis of PC12 cells were detected by Annexin-V-FITC flow cytometry.

RESULTSU0126, but not SB203580 could inhibit the activation of ERK1/2 induced by electromagnetic exposure. U0126 and SB203580 had no effects on the activation of JNK. SB203580 could inhibit the activation of P38 MAPK significantly. But U0126 had no such effect on the activation of P38 MAPK. After pretreated by SB203580 alone or together with U0126, the apoptosis of PC12 cells decreased. But the pretreatment by U0126 alone had no influence on the apoptosis of PC12 cells.

CONCLUSIONThe P38 MAPK signal transduction modulate the apoptosis of PC12 cells induced by electromagnetic exposure. ERK signal transduction has no effect on the apoptosis of PC12 cells. JNK signal transduction may promote the apoptosis of PC12 cells in the early stage after electromagnetic exposure.

Animals ; Apoptosis ; radiation effects ; Electromagnetic Radiation ; Mitogen-Activated Protein Kinases ; metabolism ; PC12 Cells ; Phosphorylation ; Rats ; Signal Transduction

OBJECTIVETo investigate the changes of ataxia-telangiectasia mutated (ATM) phosphorylation in HepG(2) cells in relation to HepG(2) cell survival under continuous low dose-rate irradiation.

METHODSHepG(2) cells were exposed to equivalent irradiation doses delivered at either a continuous low dose-rate (7.76 cGy/h) or a high dose-rate (4500 cGy/h), and the phosphorylated ATM proteins and surviving fraction of HepG(2) cells after the exposures were compared.

RESULTSThe phosphorylation of ATM protein was maximal at 0.5 Gy irradiation delivered at either a high doserate or a continuous low doserate. As the radiation dose increased, ATM protein phosphorylation decreased under continuous low dose-rate irradiation, but remained stable under high dose-rate irradiation. With comparable ATM protein phosphorylation induced by continuous low dose-rate irradiation and high dose-rate irradiation, there was no significant difference in the surviving fraction of HepG(2) cells (P>0.05), but at a significantly lower ATM protein phosphorylation level than that induced by high dose-rate irradiation, continuous low dose-rate irradiation resulted in increased cell killing (P<0.01).

CONCLUSIONContinuous low dose-rate irradiation increases HepG(2) cells radiosensitivity as compared with high dose-rate irradiation. Increased cell killing following continuous low dose-rate irradiation is associated with reduced phosphorylated ATM protein, and inhibition of ATM phosphorylation may increase the radiosensitivity of HepG(2) cells.

Animals ; Ataxia Telangiectasia Mutated Proteins ; Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Cell Survival ; radiation effects ; DNA-Binding Proteins ; metabolism ; Dose-Response Relationship, Radiation ; Humans ; Mice ; Phosphorylation ; radiation effects ; Protein-Serine-Threonine Kinases ; metabolism ; Radiation Tolerance ; radiation effects ; Time Factors ; Tumor Suppressor Proteins ; metabolism

Phosphorylation of caveolin-1 occurs during cell activation by various stimuli. In this study, the involvement of caveolin-1 in an irradiation injured spinal cord was examined by analyzing the phosphorylation of caveolin-1 in the spinal cord of rats after irradiation with a single dose of 15 Gray from a (60)Co gamma-ray source at 24 h post-irradiation (PI). A Western blot analysis showed that the phosphorylated form of caveolin-1 (p-caveolin-1) was expressed constitutively in the normal spinal cords and was significantly higher in the spinal cord of irradiated rats at 24 h PI. The increased expression of ED1, which is a marker of activated microglia/macrophages, was matched with that of p-caveolin-1. In the irradiated spinal cords, there was a higher level of p-caveolin-1 immunoreactivity in the isolectin B4-positive microglial, ependymal, and vascular endothelial cells, in which p-caveolin-1 was weakly and constitutively expressed in the normal control spinal cords. These results suggest that total body irradiation induces activation of microglial cells in the spinal cord through the phosphorylation of caveolin-1.
Animals ; Blotting, Western/veterinary ; Caveolin 1/*metabolism ; Gene Expression Regulation/*radiation effects ; Immunohistochemistry/veterinary ; Male ; Phosphorylation/radiation effects ; Rats ; Rats, Sprague-Dawley ; Spinal Cord/physiopathology/*radiation effects ; Spinal Cord Injuries/physiopathology/*veterinary