This study explored the role of radiation-induced autophagy in low-dose hyperradiosensitivity (HRS) in the human lung cancer cell line A549. A549 cells, either treated with an autophagic inhibitor 3-methyladenine (3-MA), or with a vehicle control, were irradiated at different low doses (≤0.5 Gy). The generation of autophagy was examined by laser scanning confocal microscopy. Western blotting was used to detect the expression of microtubule-associated protein l light chain 3B II (LC3B-II). Flow cytometry (FCM) and clonogenic assays were used to measure the fraction of surviving cells at the low irradiation doses. Our results showed that there was a greater inhibition of autophagic activity, but a higher degree of low-dose HRS in A549 cells treated with 3-MA than in control group. Our data demonstrated that radiation-induced autophagy is correlated with HRS in A549 cells, and is probably one of the mechanisms underlying HRS.
Adenine ; analogs & derivatives ; pharmacology ; Autophagy ; drug effects ; radiation effects ; Blotting, Western ; Cell Line, Tumor ; Cell Survival ; drug effects ; radiation effects ; Dose-Response Relationship, Radiation ; Flow Cytometry ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Microtubule-Associated Proteins ; genetics ; metabolism ; Phagosomes ; drug effects ; radiation effects ; ultrastructure ; Radiation Tolerance ; drug effects ; radiation effects

The aberrant protein crosslinks formation during lung injury as results total body irradiation (TBI) and bone marrow transplantation (BMT) therapy has been examined as apossible contributory factor in organ or tissue pathogenesis. Female C3HeB/ FeJ mice were used for an experimental animal. Carbon monoxide uptake (V(CO)) was measured at 1, 2, 3, 4 and 5 months after TBI at respective doses of 12, 14, 16 and 18 Gy 16 h prior to syngeneic BMT. Also as a measure of aberrant protein crosslinking in the inured tissues, transglutaminase (TGase)-activities and crosslinked protein were examined along with thrombin, a protease known to activate TGases. Reductions of VCO were detected following TBI and BMT. Activities of thrombin and TGase 1, and crosslinked protein in bronchoalveolar lavage (BAL) fluid of the mice 1 wk after TBI at 12 Gy and BMT were identified and found to be elevated in the treated animals. These findings suggest that elevated levels of crosslinked proteins and TGase I in the bronchoalveolar larvage during the lung injury could have enhanced the organ pathogenesis following TBI and BMT.
Animals ; *Bone Marrow Transplantation ; Carbon Monoxide/metabolism ; Factor XIII/metabolism ; Female ; Lung/*metabolism/pathology/radiation effects ; *Lung Injury ; Mice ; Proteins/*metabolism ; Thrombin/metabolism ; Transglutaminases/metabolism ; *Whole-Body Irradiation

BACKGROUND: Lung cancer is one of the highest morbidity and mortality in the world and it is very important to find an effective anti-tumor method. Microwave hyperthermia, a new treatment technology, has been getting more and more attention. This study was designed to investigate the effects of microwave hyperthermia combined with gemcitabine on the proliferation and apoptosis of human lung squamous cell carcinoma (NCI-H1703 and NCI-H2170) in vitro. METHODS: The proliferation of cells treated with microwave hyperthermia, the effect of gemcitabine on cell proliferation and the proliferation of cells treated with different methods of microwave hyperthermia and gemcitabine were detected by CCK-8 assay. Colony formation assay was used to measure the colony formation of human lung squamous cell carcinoma cells. Flow cytometry assay was used to detect the total apoptosis rates of the treated cells. Caspase-3, Caspase-8 activity assay was used to detect the activity of Caspase-3, Caspase-8 enzyme in each group of cells. CCK-8 assay was used to detect the effect of control group, AC-DEVD (Caspase-3 inhibitor) group, thermalization combined group, and thermal AC-DEVD combined group on cell proliferation. The levels of p53, Caspase-3, Cleaved-Caspase-3, PARP, Bax and BCL-2 protein expression were detected using Western blot assay. RESULTS: Our results demonstrated that microwave hyperthermia inhibited the proliferation of lung squamous cell carcinoma. The IC₅₀ values of gemcitabine for the two cells were 8.89 μmol/L and 44.18 μmol/L, respectively. The first chemotherapy after microwave hyperthermia has synergistic effect on the two lung squamous cell carcinoma cells and can significantly inhibit the cell clone formation (P<0.001), promote cell apoptosis (P<0.001) and increase Caspase-3 enzyme activity (P<0.001). However, it has no effect on Caspase-8 enzyme activity (P>0.05). Furthermore, Western blot analysis showed that microwave hyperthermia combined with gemcitabine could up-regulate the p53, Caspase-3, Cleaved-Caspase-3, Cleaved-PARP and Bax protein expression. CONCLUSIONS Microwave hyperthermia combined with gemcitabine remarkably inhibit the proliferation and induce apoptosis of human lung squamous cell carcinoma in vitro. This effect may be associated with the activation of p53, cleavage of PARP protein, and induced the Caspase-3 dependent apoptosis.
Apoptosis ; drug effects ; radiation effects ; Carcinoma, Squamous Cell ; pathology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; radiation effects ; Combined Modality Therapy ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Humans ; Hyperthermia, Induced ; Lung Neoplasms ; pathology ; Microwaves

OBJECTIVETo explore the radiosensitization effect of black garlic extract (BGE) on lung cancer cell line Lewis cells.

METHODSThe inhibition rate of lung cancer cells after BGE action was detected by MTT. Effect of BGE combined radiotherapy on the colony formation rate was observed by cloning formation assay. Changes of the cell morphology were observed by Hoechst staining. Changes of the cell cycle were detected by flow cytometry. Real time PCR was used to detect mRNA expressions of bcl-2 and bax.

RESULTSBGE could have significant inhibitory action on the growth of lung cancer Lewis cells. The combination of BGE and radiotherapy (by 60Co gamma) significantly induced Lewis cells' apoptosis in G2/M stage, obviously decreased the expression of bcl-2, and up-regulated the expression of bax.

CONCLUSIONSBGE could sensitize the lung cancer Lewis cells to ionizing irradiation. This effect might be probably caused by changing the cell cycles and affecting expressions of bax and bcl-2.

Animals ; Apoptosis ; Carcinoma, Lewis Lung ; metabolism ; pathology ; Cell Line, Tumor ; drug effects ; radiation effects ; Garlic ; Mice ; Plant Extracts ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Radiation-Sensitizing Agents ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the relationship between epidermal growth factor receptor (EGFR) gene expression and radiosensitivity of non-small-cell lung cancer (NSCLC) cells.

METHODSEGFR sequence-specific double-stranded RNA (dsRNA-EGFR) was chemically synthesized. NSCLC cell line SPC-A1 was transfected with dsRNA-EGFR formulated with Lipofectamine 2000. Western blot and real-time PCR were used to determine the EGFR mRNA and protein expression, respectively. Colony inhibition test was adopted to observe the radiosensitizing effect. To establish the nude mouse tumor models, calculate the tumor growth inhibition rate and make the tumor growth curve by measuring its size and weight.

RESULTSEGFR mRNA levels were 1.51 ± 0.22, 1.38 ± 0.15 and 0.45 ± 0.11 in the control group, dsRNA-unrelated group and dsRNA-EGFR group, respectively (F = 482.7, P < 0.01). The contents of EGFR protein were 2340.87 ± 10.99, 2231.85 ± 35.66 and 832.03 ± 39.13 in the control group, dsRNA-unrelated group and dsRNA-EGFR group, respectively (F = 263.3, P < 0.05). Compared with the control group, dsRNA-EGFR sequence specifically decreased the expressions of EGFR mRNA by 70.2% and EGFR protein by 64.5%. The colony inhibition rates of the control group, dsRNA-unrelated combined with radiotherapy group and dsRNA-EGFR combined with radiotherapy group were 9.3%, 12.5% and 65.5%, and the tumor growth inhibition rates were 21.3%, 24.4% and 64.2%, respectively. The combination of dsRNA-EGFR and radiotherapy significantly inhibited the tumor growth in vitro and in vivo.

CONCLUSIONSDsRNA-EGFR shows an apparent inhibitory effect on the expression of EGFR mRNA and protein of NSCLC cells, effectively inhibit the tumor growth in vivo, and enhance the radiosensitivity of NSCLC.

Adenocarcinoma ; metabolism ; pathology ; radiotherapy ; Animals ; Cell Line, Tumor ; Cell Proliferation ; radiation effects ; Humans ; Lung Neoplasms ; metabolism ; pathology ; radiotherapy ; Male ; Mice ; Mice, Nude ; Neoplasm Transplantation ; RNA, Double-Stranded ; genetics ; RNA, Messenger ; metabolism ; Radiation Tolerance ; Random Allocation ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Transfection ; Tumor Burden ; radiation effects

OBJECTIVETo compare the treatment results of three-dimensional conformal radiotherapy (3D-CRT) and conventional radiotherapy (2D) for patients with locally advanced non-small-cell lung cancer (NSCLC).

METHODSFive hundred and twenty seven patients with stage III NSCLC treated between Jan 2000 and Dec 2006 were included in this study. Among them, 253 cases were treated with 3D-CRT, and 274 with conventional radiotherapy. In the 3D group, 159 (62.8%) patients received chemoradiotherapy, 77 with total radiotherapy dose of > 60 Gy, 49 with 50 - 60 Gy. In the 2D group, 127 (46.4%) patients received chemoradiotherapy, 48 with total radiotherapy dose of > 60 Gy, 75 with 50 - 60 Gy.

RESULTSThe 1-, 3-, 5-year overall survival rates (OS) and median survival time for patients treated with 3D-CRT were 73.3%, 26.1%, 14.4% and 20.1 months, respectively, and that of patients treated with 2D radiotherapy were 61.0%, 13.8%, 8.0% and 15.6 months, respectively (P = 0.002). The 1-, 3-, 5-year cause-specific survival rates (CSS) were 79.0%, 33.3%, and 20.8% for the 3D group and 65.1%, 16.7%, 11.2%, respectively, for the 2D group (P = 0.000). The 1-, 3-, and 5-year locoregional control rates were 71.6%, 34.3% and 31.0% for patients treated with 3D radiotherapy and 57.3%, 22.1% and 19.2%, respectively, for patients treated with 2D treatment (P = 0.002). The results of multivariate analysis showed that 3D-CRT, KPS, clinical tumor response and pretreatment hemoglobin level were independently associated with increased OS and CSS. No statistically significant differences were found between the radiation complications in the two groups.

CONCLUSIONSThe results of our study demonstrate that 3D-conformal radiotherapy improves the survival rate in patients with stage III NSCLC compared with that of 2D radiation therapy.

Aged ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; pathology ; radiotherapy ; Chemoradiotherapy ; Female ; Follow-Up Studies ; Hemoglobins ; metabolism ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; radiotherapy ; Male ; Neoplasm Staging ; Radiation Pneumonitis ; etiology ; Radiotherapy Dosage ; Radiotherapy, Conformal ; adverse effects ; methods ; Survival Rate

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

AIMTo study the effects of bone marrow MSCs transplantation on the apoptosis of alveolar wall cells and the expression of Bcl-2 and Bax of lung tissue in papain and Co60-induced pulmonary emphysema rats.

METHODSFemale Lewis rats were randomly divided into three groups: control group, emphysema group, emphysema + MSCs transplantation group. Rats were sacrificed at days 14 and 28 after treatment. Morphologic analysis of the lung tissue was performed. The apoptosis of the lung cells was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of Bcl-2 and Bax were determined by immunohistochemical staining.

RESULTSEmphysematous changes of the lung tissue were observed in emphysema group and emphysema + MSCs transplantation group. However, the emphysematous change in emphysema + MSCs transplantation group was improved compared with the emphysema group. There was significant difference in the number of alveolar counted per unit area (MAN), mean alveoli area (MAA) and mean linear interval(MLI) between emphysema group and emphysema + MSCs transplantation group. The apoptotic index of the alveolar wall cells in emphysema + MSCs transplantation group was less than that in the emphysema group. The percentage of Bcl-2 positive cells in emphysema + MSCs transplantation group was significantly higher than that in the emphysema group. The percentage of Bax positive cells in emphysema + MSCs transplantation group was significantly lower than that in the emphysema group. The ratio of Bcl-2/Bax of emphysema + MSCs transplantation group was significantly higher than that in the emphysema group.

CONCLUSIONBone marrow MSCs transplantation inhibits the apoptosis of alveolar wall cells, upregulates the expression of Bcl-2 and downregulates the expression of Bax. This may be part of the reason that bone marrow MSCs transplantation improves the papain and Co60-induced pulmonary emphysema.

Animals ; Apoptosis ; Bone Marrow Transplantation ; Cells, Cultured ; Cobalt Isotopes ; adverse effects ; Female ; Lung ; cytology ; Mesenchymal Stem Cell Transplantation ; Papain ; adverse effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pulmonary Alveoli ; drug effects ; pathology ; radiation effects ; Pulmonary Emphysema ; chemically induced ; metabolism ; pathology ; surgery ; Rats ; bcl-2-Associated X Protein ; metabolism