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

BACKGROUNDPerineural invasion (PNI) is a histopathological characteristic of pancreatic cancer (PanCa). The aim of this study was to observe the treatment effect of continuous low-dose-rate (CLDR) irradiation to PNI and assess the PNI-related pain relief caused by iodine-125 ( 125 I) seed implantation.

METHODSThe in vitro PNI model established by co-culture with dorsal root ganglion (DRG) and cancer cells was interfered under 2 and 4 Gy of 125 I seeds CLDR irradiation. The orthotopic models of PNI were established, and 125 I seeds were implanted in tumor. The PNI-related molecules were analyzed. In 30 patients with panCa, the pain relief was assessed using a visual analog scale (VAS). Pain intensity was measured before and 1 week, 2 weeks, and 1, 3, and 6 months after 125 I seed implantation.

RESULTSThe co-culture of DRG and PanCa cells could promote the growth of PanCa cells and DRG neurites. In co-culture groups, the increased number of DRG neurites and pancreatic cells in radiation group was significantly less. In orthotopic models, the PNI-positive rate in radiation and control group was 3/11 and 7/11; meanwhile, the degrees of PNI between radiation and control groups was significant difference (P < 0.05). At week 2, the mean VAS pain score in patients decreased by 50% and significantly improved than the score at baseline (P < 0.05). The pain scores were lower in all patients, and the pain-relieving effect was retained about 3 months.

CONCLUSIONSThe CLDR irradiation could inhibit PNI of PanCa with the value of further study. The CLDR irradiation could do great favor in preventing local recurrence and alleviating pain.

Animals ; Apoptosis ; radiation effects ; Cell Line, Tumor ; Coculture Techniques ; Dose-Response Relationship, Radiation ; Ganglia, Spinal ; cytology ; Humans ; Iodine Radioisotopes ; therapeutic use ; Mice ; Mice, SCID ; Neoplasm Recurrence, Local ; radiotherapy ; Pancreatic Neoplasms ; radiotherapy ; Rats

OBJECTIVETo explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest.

METHODSProtein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence- associated-β-galactosidase (SA-β-gal) staining combined with Ki67 staining, a cell proliferation marker.

RESULTSAccompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells.

CONCLUSIONOur results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis.

Cell Cycle Checkpoints ; radiation effects ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; DNA Damage ; Down-Regulation ; Fibroblasts ; metabolism ; radiation effects ; Gene Expression Regulation ; radiation effects ; Humans ; Mitosis ; radiation effects ; RNA Interference ; RNA, Small Interfering ; Radiation, Ionizing ; Up-Regulation

OBJECTIVE: To explore the effects of 3-methyladenine (3-MA, an autophagy inhibitor) on sensitivities of nasopharyngeal carcinoma cells to radiotherapy and chemotherapy and the underlying mechanisms.
 METHODS: Cell proliferation was examined by MTT and colony formation assay, while cell apoptosis was evaluated by annexin V/PI double staining and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. Mitochondrial membrane potential was measured by commercial kit (JC-1). The expression of endoplasmic reticulum stress (ERS)-related protein, glucose-regulated protein 78 (GRP78) and autophagy-related protein beclin1, microtubule-associated protein 1 light chain 3 (LC3) were examined by Western blot.
 RESULTS: Cisplatin (DDP), ionizing radiation (IR) or tunicamycin (TM) treatment obviously inhibited the proliferation of HONE-1 cells in a concentration-dependent and time-dependent manner. Compared with control group, pretreatment with 1 mmol/L of 3-MA significantly 
reduced cell viability and enhanced the apoptosis in the DDP (6.00 μmol/L), 4.00 Gy IR or TM (1.00 μmol/L) groups. There was no significant difference in the apoptosis between the DDP (5.8%) and 4Gy IR (6.7%) groups. Compared with the control group, protein levels of GRP78, beclin1 and lipid-conjugated membrane-bound form (LC3-II) were significantly increased after the treatment of DDP, 4.00 Gy IR or TM, which were inhibited by pretreatment of 3-MA.
 CONCLUSION 3-MA can sensitize HONE-1 cells to chemotherapy and radiotherapy, which is related to prevention of endoplasmic reticulum stress-induced autophagy in nasopharyngeal carcinoma cells.
Adenine ; analogs & derivatives ; pharmacology ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Beclin-1 ; Carcinoma ; Cell Line, Tumor ; drug effects ; radiation effects ; Cell Proliferation ; Cell Survival ; Cisplatin ; pharmacology ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; Membrane Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; pathology ; Radiation, Ionizing ; Radiation-Sensitizing Agents ; pharmacology ; Tunicamycin ; pharmacology

OBJECTIVETo study the radiosensitizing effect of resveratrol on hypopharyngeal carcinoma cell line FADU in vitro.

METHODSHypopharyngeal carcinoma cell line FADU was cultured in in vitro DMEM. Its inhibition on cell proliferation was detected using cytotoxicity test (MTT assay). The cell survival curve was drawn using clone formation to obtain sensitive enhancement ratio (SER). Changes of the cell cycle and cell apoptosis were analyzed using flow cytometry (FCM).

RESULTSResults of MTT showed the inhibition of resveratrol on FADU cells increased along with its concentrations (P < 0.05). Results of clone formation indicated the surviving fraction at 2 Gy (SF2) was 0.717 ± 0.062 in the irradiation group, and 0.426 ± 0.035 in the resveratrol plus irradiation group (with SER ranged 1.684 ± 0.178) with statistical difference (P = 0.007). Results of FCM showed that after radiation of 4 Gy radiation, cells at G2/M phase arrest increased, but cells at G1 decreased. After radiation of resveratrol for 24 h, cells at G1 decreased, but cells at G2/M phase and S phase arrest increased. When 4 Gy radiation combined resveratrol was used, cells at G2/M phase arrest significantly increased, but cells at G1 significantly decreased. The apoptosis rate was 1.94% ± 1.65% in the control group, 4.56% ± 0.92% in the irradiation group, 2.03% ± 1.46% in the resveratrol group, and 23.11% ± 7.22% in the resveratrol plus irradiation group. There was statistical difference between the resveratrol plus irradiation group and the rest 3 groups (P < 0.05).

CONCLUSIONResveratrol could enhance the radiosensitivity of hypopharyngeal carcinoma FADU cells in vitro possibly by inducing cell apoptosis and causing changes in the cell cycle distribution.

Apoptosis ; Carcinoma, Squamous Cell ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Head and Neck Neoplasms ; Humans ; Hypopharyngeal Neoplasms ; drug therapy ; Radiation Tolerance ; Radiation-Sensitizing Agents ; therapeutic use ; Stilbenes ; therapeutic use

OBJECTIVETo investigate the effect of RNA interference of IgG gene on the radiosensitivity of the human prostate cancer PC3 cell line.

METHODSPC3 cells were trasnfected via lipofectamine by the shRNA vector FCGR1AshRNA targeting the Fc segment of IgG, using NCshRNA as the negative control. Q-PCR and Western blotting were used to analyze the expression of IgG in the trasnfected cells. The cells were then exposed to ⁶⁰Co γ ray at 0, 2, 4, 6, 8, 10 Gy, and the cell proliferation was evaluated by MTS and the cells apoptosis estimated by flow cytometry at 12, 24 and 48 h.

RESULTSMTS assay showed that ⁶⁰Co γ ray significantly inhibited the proliferation of PC3 cells transfected with FCGR1AshRNA as compared with NCshRNA-transfected and blank control cells (P<0.05). Flow cytometry showed that the cell apoptosis rate was significantly higher in FCGR1AshRNA group than in NCshRNA and blank control groups at 48 h after γ ray exposure (P<0.05). At 12, 24 and 48 h after 6 Gy radiation, the cells in FCGR1AshRNA group showed a significantly lowered proliferation rate and an increased apoptosis rate (P<0.05).

CONCLUSIONThe shRNA targeting IgG gene can significantly enhance the sensitivity of PC3 cells to radiation. The combination of RNA interference targeting IgG gene with radiotherapy may be more effective in the treatment of prostate cancer.

Apoptosis ; Cell Line, Tumor ; radiation effects ; Cell Proliferation ; Humans ; Immunoglobulin G ; genetics ; Male ; Prostatic Neoplasms ; pathology ; RNA Interference ; RNA, Small Interfering ; Radiation Tolerance ; Transfection

BACKGROUNDCaffeine suppresses ataxia telangiectasia and Rad3 related and ataxia telangiectasia mutated (ATM) activities; ATM is the major kinase for DNA damage detection. This study aimed to investigate the effects of caffeine on DNA damage responses in cells from the bladder cancer cell line RT4 those were exposed to ionizing radiation (IR).

METHODSImmunofluorescent staining was performed to investigate changes in the proteins involved in DNA damage responses with or without caffeine. A mouse xenograft model was used to study the effects of caffeine on the DNA damage responses. Western blotting was used to investigate the effects of caffeine pretreatment on the ATM-Chk2-p53-Puma axis, while real-time polymerase chain reaction (RT-PCR) assessed changes in messenger RNA levels of p53 and downstream targets responding to IR. Finally, terminal deoxynucleotidyl transferase-dUTP nick end labeling assay. Western blotting and colony formation assay were used to measure the effects of caffeine on radiation-related apoptosis. All of the data were analyzed with a two-tailed Student's t-test.

RESULTSImmunofluorescent staining showed that caffeine pretreatment profoundly suppressed the formation of γH2AXand p53-binding protein 1 foci in RT4 cells in response to irradiation. Cellular and animal experiments suggested that this suppression was mediated by suppression of the ATM-Chk2-p53-Puma DNA damage-signaling axis. RT-PCR indicated caffeine also attenuated transactivation of p53 and p53-inducible genes. The colony formation assay revealed that caffeine displayed radioprotective effects on RT4 cells in response to low-dose radiation compared to the radiosensitization effects on T24 cells.

CONCLUSIONCaffeine may inhibit IR-related apoptosis of bladder cancer RT4 cells by suppressing activation of the ATM-Chk2-p53-Puma axis.

Animals ; Apoptosis ; drug effects ; radiation effects ; Caffeine ; pharmacology ; Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Checkpoint Kinase 2 ; metabolism ; Humans ; Immunohistochemistry ; Male ; Mice ; Mice, Nude ; Radiation, Ionizing ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Tumor Suppressor Protein p53 ; metabolism ; Urinary Bladder Neoplasms ; radiotherapy

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

OBJECTIVE: To study the relationship between the radiotherapy resistance and autophagy. To provide a theoretiacal basis for drugs that regulate autophagy to improve radiotherapy sensitivity. METHOD: Flow cytometry (FCM) was performed to analyze the distribution of the cell cycle of CNE2 and CNE2/DDP cells under the action of X radiation. The expression of autopagy-specific gene Beclin1 and microtubule-associated protein light chain 3β (MAPLC3β) in CNE2 and CNE2/DDP cells was determined by real time PCR and Immumofluorescence staining. RESULT: CNE2/DDP and their parental CNE2 cells produced the G2-M phase arrest under the action of X radiation. With the radiation dose increasing,The cells which in the G2-M phase were more and more (P<0. 05). The G2-M phase arrest in CNE2/DDP cells was more obvious than in CNE2 cells (P<0. 05). The expression of Beclin1 and MAPLC3β in CNE2 and CNE2/DDP cells increased under the action of X radiation. What's more, the raise was more and more obvious with the increase of the irradiation dose(P<0. 05). The expression levels of Beclin1 and MAPLC3β in CNE2/DDP was lower than that in CNE2 cells (P<0. 05). CONCLUSION Autophagic cell death may be the one manner of death in nasopharyngeal carcinoma CNE2 and CNE2/DDP cells under the action of X radiation. The radiation resistance of CNE2/DDP cells may be related to the low expression of autophagy-related genes.
Apoptosis Regulatory Proteins ; genetics ; Autophagy ; Beclin-1 ; Carcinoma ; Cell Cycle ; Cell Line, Tumor ; radiation effects ; Dose-Response Relationship, Radiation ; Humans ; Membrane Proteins ; genetics ; Microtubule-Associated Proteins ; genetics ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; Radiation Tolerance ; X-Rays

OBJECTIVETo study the radiosensitizing effect of resveratrol on human hypo pharyngeal squamous cell carcinoma (FaDu) cells in nude mice.

METHODSForty-three nude mice bearing FaDu cell xenografts were randomized into control group, radiotherapy (12 Gy) group, resveratrol treatment (50 mg/kg) group, and radiotherapy plus resveratrol treatment group. After corresponding treatments, the tumor volume in the mice was measured every 3 days, and the microvessel density (MVD) in the tumor was evaluated with CD31 immunofluorescence histochemical staining.

RESULTSThe tumor volume and weight were the smallest in mice receiving radiotherapy plus resveratrol treatment (P<0.05) but comparable between those having resveratrol treatment alone and the control mice. Radiotherapy plus resveratrol treatment resulted in a tumor inhibition rate of 76.64% and a significantly decreased MVD in the tumor compared with the other 3 groups.

CONCLUSIONResveratrol can produce a radiosensitizing effect on human hypopharyngeal carcinoma in nude mice.

Animals ; Carcinoma, Squamous Cell ; drug therapy ; radiotherapy ; Cell Line, Tumor ; radiation effects ; Head and Neck Neoplasms ; drug therapy ; radiotherapy ; Humans ; Hypopharyngeal Neoplasms ; drug therapy ; radiotherapy ; Mice ; Mice, Nude ; Radiation Tolerance ; Radiation-Sensitizing Agents ; pharmacology ; Stilbenes ; pharmacology ; Transplantation, Heterologous ; Tumor Burden