Expression of active caspase 3 in the rat lens after in vivo exposure to subthreshold dose of UVR-B.

PURPOSES: The aim of this study is to investigate the time evolution of active caspase 3 within first 120 h in the rat lens after in vivo exposure to subthreshold dose of UVR-B. METHODS: Twenty three six-week-old female albino Sprague-Dawley rats were exposed to subthreshold dose (1 kJ/m2) of UVR-B unilaterally and sacrificed at 24, 41, 70 and 120 h after exposure. Lenses were enucleated and active caspase 3 was detected by Western Blot. The time evolution of active caspase 3 was then plotted as a function of relative mean difference in active caspase 3 between exposed and nonexposed lenses. RESULTS: There is expression of active caspase 3 in both exposed and nonexposed lenses but there is no difference in relative mean difference in active caspase 3 between exposed and nonexposed lenses in all four postexposure groups. CONCLUSIONS: Exposure to subthreshold dose of UVR-B does not induce apoptosis in the rat lens in vivo within first 120 h though there is a non-significant increase of active caspase 3 at 120 h. Increase in sample size might reduce the variation level in expression of active caspase 3 in the rat lenses.

Thymoquinone Enhances the Effect of Gamma Knife in B16-F10 Melanoma Through Inhibition of Phosphorylated STAT3.

BACKGROUND: Patients with brain metastasis from melanoma have a dismal prognosis with poor survival time. Gamma Knife (GK) is an effective treatment to control brain metastasis from melanoma. Thymoquinone (TQ) has emerged as a potential therapeutic option due to its antiproliferative effects on various cancers. The purpose of the study was to assess the effect of GK on B16-F10 melanoma cells in vitro and intracerebral melanoma in vivo, and its synergistic effect in combination with TQ. METHODS: The effects of GK and combination treatment of GK and TQ were studied on B16-F10 melanoma cells by evaluating cytotoxicity with an adenosine triphosphate assay, apoptosis by acridine orange staining, and genotoxicity by comet assay. Western blot analysis was performed to investigate the expression of STAT3, p-STAT3 (Tyr705), JAK2, p-JAK2, caspase-3, Bax, Bcl-2, survivin, and ß-actin. Expression of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. GK alone and in combination with TQ was assessed in an established intracerebral melanoma tumor in mice. RESULTS: The effects of GK on cytotoxicity, genotoxicity, and apoptosis were enhanced by TQ in B16-F10 melanoma cells. GK induced apoptosis through inhibition of p-STAT3 expression, which in turn regulated pro- and antiapoptotic proteins such as caspase-3, Bax, Bcl-2, and survivin. Adding TQ to GK irradiation further enhanced this apoptotic effect of GK irradiation. GK was shown to reduce the levels of tumor-related inflammatory cytokines in B16-F10 melanoma cells. This effect was more pronounced when TQ was added to GK irradiation. GK with 15 Gy increased the survival of mice with intracerebral melanoma compared with untreated mice. However, despite the additive effect of TQ in addition to GK irradiation on B16-F10 melanoma cells in vitro, TQ did not add any significant survival benefit to GK treatment in mice with intracerebral melanoma. CONCLUSIONS: Our findings suggest that TQ would be a potential therapeutic agent in addition to GK to enhance the antitumor effect of irradiation. Further studies are required to support our findings.

ROS Induced by KillerRed Targeting Mitochondria (mtKR) Enhances Apoptosis Caused by Radiation via Cyt c/Caspase-3 Pathway.

During radiotherapy, reactive oxygen species- (ROS-) induced apoptosis is one of the main mechanism of radiation. Based on KillerRed which can induce ROS burst in different cell substructures, here we hypothesized that KillerRed targeting mitochondria (mtKR) could induce ROS to enhance apoptosis by radiation. In this study, empty vector, mtKR, and mtmCherry plasmids were successfully constructed, and mitochondrial localization were detected in COS-7 and HeLa cells. After HeLa cells were transfected and irradiated by visible light and X-rays, ROS levels, mitochondrial membrane potential (Δψ m), ATPase activities, adenosine triphosphate (ATP) content, apoptosis, and the expressions of mRNA and protein were measured, respectively. Data demonstrated that the ROS levels significantly increased after light exposure, and adding extra radiation, voltage-dependent anion channel 1 (VDAC1) protein increased in the mitochondria, while Na+-K+ and Ca2+-Mg2+ ATPase activities, ATP content, and Δψ m significantly reduced. Additionally, the cell apoptotic rates dramatically increased, which referred to the increase of cytochrome c (Cyt c), caspase-9, and caspase-3 mRNA expressions, and Cyt c protein was released from the mitochondria into the cytoplasm; caspase-9 and -3 were activated. These results indicated that mtKR can increase the production of ROS, enhance mitochondrial dysfunction, and strengthen apoptosis by radiation via Cyt c/caspase-3 pathway.

Phenylephrine Alleviates 131I Radiation Damage in Submandibular Gland Through Maintaining Mitochondrial Homeostasis.

PURPOSE: The impairment of the salivary glands is a permanent side effect of 131I ablation therapy for patients with differentiated thyroid cancer. Effective and safe treatments for protecting the salivary glands against 131I are currently not available. Mitochondria are susceptible to ionizing radiation, but alterations after 131I exposure are unknown. Here, we investigated the mechanisms of 131I damage in submandibular glands (SMGs) and evaluated the cytoprotective effect of phenylephrine (PE) against mitochondrial radiation damage. METHODS AND MATERIALS: Rats were randomly divided into 4 groups: control, PE alone, 131I alone, and 131I with PE pretreatment. The mitochondrial structure of SMGs was observed under transmission electron microscopy. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Cytochrome c, cleaved-caspase 3, SIRT1, NAMPT, and PGC-1α protein levels were determined with Western blot and immunohistochemistry. Levels of mitochondrial membrane potential, nicotinamide adenine dinucleotide (NAD), and adenosine triphosphate (ATP) were measured with relevant kits. RESULTS: After exposing rat SMGs to 131I, the mitochondrial membrane structures were destroyed, the mitochondrial membrane potential decreased, the release of cytochrome c increased, and cleaved-caspase 3 and cell apoptosis were activated. Moreover, the expression of SIRT1, NAMPT, and PGC-1α was downregulated, and the levels of NAD and ATP decreased. In contrast, PE alleviated the 131I-induced mitochondrial damages and upregulated the expression of SIRT1/NAMPT/PGC-1α and the levels of NAD and ATP. CONCLUSIONS: These findings demonstrate that 131I impairs the salivary glands via the downregulation of SIRT1/NAMPT/PGC-1α signal pathways, which disturbs mitochondrial homeostasis. PE alleviated the 131I damage in SMGs at the mitochondrial level, suggesting that PE could be used as a potential radioprotector for patients with differentiated thyroid cancer with radiation sialadenitis.

[Effect of high frequency electrotherapy on caspase-3 and ultra microstructure of hippocampus in rats following cerebral ischemia/reperfusion].

OBJECTIVE: To investigate the effect of high frequency electrotherapy (HFE) on rat hippocampus after cerebral ischemia/reperfusion (I/R).
 Methods: A rat model of cerebral I/R injury was established. The rats were randomly divided into a sham group, an I/R group and an HFE group. The HFE group received thearapy daily for different sessions for 1, 3, 7 d. Neuronal deficit score,neuron ultra microstructure in the hippocampus and caspase-3 protein expression were measured on 1 st, 3 th and 7th d.
 Results: Compared with the I/R group, the HFE group showed the decreased neurological deficit scores, with significant differences between the 2 groups (P<0.05). The injury in HFE group was reduced compared with that in the I/R group based on the electron microscope test, with significant difference. Caspase-3 protein in brain tissue in the HFE group also downregulated compared with that in the I/R group (P<0.05).
 Conclusion: High frequency electrotherapy can improve neural function, suppress caspase-3 expression and apoptosis in nerve cells and improve the ultra microstructure of neurons, displaying a protective effect on cerebral I/R injury in rats.

[The cardiac injury effect of microwave radiation on rabbit and its mechanism].

OBJECTIVE: To investigate the cardiac injury effect of different intensities microwave radiation on rabbits and its possible mechanism. METHODS: Rabbits were radiated by intensity of 50, 100, 150 and 200 mW/cm2 2450 MHz microwave for 20 min. 6 h after microwave radiation, the heart tissue was taken. ATP and mitochondria complex IV and V were measured in myocardial cells. The changes of myocardial tissue were observed by light microscopic. The expression of Caspase-3 and HSP 70 were detected by western blotting. RESULTS: The activity of ATP and mitochondria complex IV and V decreased significantly compared with normal control in cardiac tissue. 100, 150 and 200 mW/cm2 microwave radiation group vs. control group (P <0. 05). The HE staining result showed that myocardial cell appears edema, muscle fiber malalignment, cells appeared obvious injury. Results of western blotting showed that the expression of Caspase-3 and HSP 70 protein increased significantly in different dosage radiation group (P <0. 05). CONCLUSION: Microwave radiation has injury effect on rabbit heart. The possible mechanism may be related with inducing cell apoptosis by changing of stress level in myocardial cell.

Optimization of a Stable Linker Involved DEVD Peptide-Doxorubicin Conjugate That Is Activated upon Radiation-Induced Caspase-3-Mediated Apoptosis.

The current study demonstrates the process of selecting an optimal structure for a caspase-3-cleavable doxorubicin prodrug that could be synthesized by simple chemistry in high yields. The prodrug was intended to activate in the presence of caspase-3, whose expression can be exogenously regulated by inducing apoptosis with radiation therapy at a specific site of interest. For this purpose, doxorubicin was conjugated with a DEVD peptide via a heterobifunctional linker. Since the active form of the prodrug comprises the linker besides doxorubicin, we tested several different linkers and selected EMCS based on the examination of its in vitro biological activities. Consequently, DEVD-cysteamide-EMCS-doxorubicin was synthesized as the final compound. According to the various in vitro and in vivo studies, the synthesized prodrug was highly selective for tumors when coupled with radiation therapy, with the added benefit of ease of production.

Carcinostatic effects of platinum nanocolloid combined with gamma irradiation on human esophageal squamous cell carcinoma.

AIMS: To explore the carcinostatic effects of platinum nanocolloid (Pt-nc) combined with gamma rays on human esophageal squamous cell carcinoma (ESCC). MAIN METHODS: ESCC-derived KYSE-70 cells were treated with various concentrations of Pt-nc and/or gamma irradiation, and subsequently cultured in phenol red free DMEM with 10% FBS for 48 h. The proliferative status of the KYSE-70 cells was evaluated using trypan blue dye exclusion and WST-8 assays. Cellular and nucleic morphological aspects were evaluated using crystal violet and Hoechst 33342 stainings, respectively. Radiosensitivity was quantified by a cell viability assay, and the activated form of caspase-3, a characteristic apoptosis-related protein, was detected by Western blotting. KEY FINDINGS: Although single treatment with either Pt-nc or gamma irradiation could slightly inhibit the growth of the KYSE-70 cells, their combination exerted remarkable carcinostatic effects in a manner dependent on either Pt-nc concentrations or gamma ray doses, compared with the effect of each treatment alone (p<0.05). By fluorescence micrographic observation, the KYSE-70 cells that were treated with Pt-nc and subsequently irradiated with gamma rays, were shown to undergo distinct apoptotic morphological changes. The carcinostatic effect of gamma rays at 7 Gy without Pt-nc was approximately equal to that when 3-Gy irradiation was combined with 100 ppm Pt-nc or that 5-Gy irradiation was combined with 50 ppm Pt-nc. SIGNIFICANCE: Pt-nc in combination with gamma rays may exert a cooperative effect through platinum- or gamma ray-induced apoptosis resulting in the inhibition of growth of cancer cells, while concurrently enabling the lowering of the radiative dose.

Radiosensitization effect of overexpression of adenovirus-mediated SIRT6 on A549 non-small cell lung cancer cells.

OBJECTIVE: To explore the radiosensitization effect of overexpression of silent information regulator 6 (SIRT6) on A549 non-small cell lung cancer (NSCLC) cells. METHODS: Adenovirus vector Ad-SIRT6 causing overexpression of SIRT6 was established. Western blotting and MTT assay were adopted to detect the level of SIRT6 protein and the inhibitory rate of A549 cell proliferation after different concentrations of adenovirus transduction (0, 25, 100, 200, and 400 pfu/cell) for 24 h. Control group, Ad-null group and Ad-SIRT6 group were designed in this experiment and virus concentration of the latter two groups was 200 pfu/cell. Colony formation assays were employed to test survival fraction (SF) of the 3 groups after 0, 2, 4, 6, 8, 10 X-ray irradiation. Flow cytometry was used to detect the status of cell cycle of 3 groups after 48 h of 4 Gy X-ray irradiation and Western blotting was used to determine the expression of apoptosis-related genes of 3 groups after 48 h of 4 Gy X-ray irradiation. RESULTS: In the range of 25 ~ 400 pfu/cell, the inhibitory rate of A549 cell proliferation increased as adenovirus concentration raised. The inhibitory rates under the concentrations of 0, 25, 100, 200, and 400 pfu/cell were 0%, 4.23 ± 0.34%, 12.7 ± 2.57%, 22.6 ± 3.38%, 32.2 ± 3.22%, 38.7 ± 4.09% and 47.8 ± 5.58% and there were significantly differences among groups (P < .05). SF in Ad-SIRT6 group was lower than Ad-null and control groups after 4 ~ 10 Gy X-ray irradiation (P < 0.05) and the sensitization enhancement ratio (SER) was 1.35 when compared with control group. Moreover, after 48 h of 4 Gy X-ray irradiation, there appeared a significant increase in G1-phase cell proportion, up-regulated expression of the level of apoptosis-promoting genes (Bax and Cleaved caspase-3), but a obvious decline in S-phase and G2-phase cell proportion and a significant decrease of the level of apoptosis- inhibiting gene (Bal-2) in the Ad-SIRT6 group (P<0.05). CONCLUSION: The over-expression of adenovirus-mediated SIRT6, which has radiosensitization effect on A549 cells of NSCLC, can inhibit the proliferation of A549 cells and cause G0/G1 phase retardation as well as induce apoptosis of cells.

Cell fate regulated by nuclear factor-κB- and activator protein-1-dependent signalling in human melanocytes exposed to ultraviolet A and ultraviolet B.

BACKGROUND: Ultraviolet (UV) radiation constitutes an important risk factor for malignant melanoma, but the wavelength responsible for the initiation of this disease is not fully elucidated. Solar UV induces multiple signalling pathways that are critical for initiation of apoptotic cell death as a cellular defence against malignant transformation. OBJECTIVES: To evaluate the involvement of the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1 in the signalling pathways induced by UVA or UVB irradiation in human melanocytes. METHODS: Primary cultures of normal human melanocytes were irradiated with UVA or UVB, and the concomitant DNA damage and redox alterations were monitored. The resulting activation of the NF-κB and AP-1 signalling pathways and subsequent apoptosis were studied. RESULTS: UVB irradiation causes DNA damage detected as formation of cyclobutane pyrimidine dimers, while UVA induces increased levels of 8-hydroxydeoxyguanosine and lipid peroxidation. UVA and UVB initiate phosphorylation of c-Jun N-terminal protein kinase and extracellular signal-regulated kinase, and the apoptosis signalling pathways converge into a common mechanism. Downregulation of c-Jun suppresses AP-1-mediated signalling and prevents apoptosis upstream of lysosomal and mitochondrial membrane permeabilization, whereas inhibition of NF-κB by SN50 increases apoptosis. CONCLUSIONS: We conclude that AP-1 induces proapoptotic signalling, whereas NF-κB is a key antiapoptotic/prosurvival factor in both UVA- and UVB-induced cellular damage in human melanocytes, which might in turn impact melanoma development and progression.

Injury in Minipig Parotid Glands following Fractionated Exposure to 30 Gy of Ionizing Radiation.

OBJECTIVE: To explore the effects of 30 Gy of (60)Co γ-rays on apoptosis and reactive oxygen species (ROS) levels in minipig parotid cells as a possible mechanism for radiation-induced parotid injury. STUDY DESIGN: Experimental study. SETTING: Department of Radiotherapy, First Affiliated Hospital, Guangxi Medical University, Nanning, China. SUBJECTS AND METHODS: Forty male minipigs were divided into control and irradiated groups. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling was used for detecting apoptosis in the parotid cells, immunohistochemistry, and western blots were used to test expression of the B-cell lymphoma 2 (Bcl-2) and BCL2-associated X (Bax) proteins, and reverse transcription polymerase chain reaction was used to analyze the expression of Bcl-2, Bax, p53, and caspase-3 messenger ribonucleic acid. An enzyme-linked immunosorbent assay was used to detect ROS levels in the parotid tissue. RESULTS: At each time point, the apoptotic rates in the irradiated group were higher than those in the control group. Furthermore, the ROS and expression levels of Bax, p53, and caspase-3 messenger ribonucleic acid and proteins gradually increased and were higher than those in the control group. Conversely, the expression of Bcl-2 was decreased in the irradiated group (P < .05). CONCLUSIONS: Ionizing radiation induces the production of ROS and promotes changes in the expression of several apoptotic proteins, which increases apoptosis and likely contributes to the mechanism of radiation-induced parotid injury.

Radiation/paclitaxel treatment of p53-abnormal non-small cell lung cancer xenograft tumor and associated mechanism.

BACKGROUND: Mutations in key tumor suppressor genes such as tumor protein 53 (TP53) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) are the main genetic alterations in cancers. TP53 mutations have been found in most patients with non-small cell lung cancer (NSCLC), whereas PTEN mutations are rarely found in lung cancer, though most NSCLCs lack PTEN protein synthesis. However, the signaling involved in radio- and chemotherapy of NSCLC with wild-type PTEN and nonfunctional p53 is not clearly understood. METHODS: In this study, we established a xenograft tumor model with H358 NSCLC cells expressing wild-type PTEN, but nonfunctional p53. Protein expression and phosphorylation of PTEN and its downstream signal molecules in NSCLC tissues were detected by Western blot. RESULTS: We demonstrated that radiation and paclitaxel alone inhibited tumor growth, but a combined therapy of radiation and paclitaxel was more effective in inhibiting NSCLC tumor growth. Interestingly, both radiation and paclitaxel significantly increased PTEN protein expression and phosphorylation. Further identification of the affected PTEN downstream molecules showed that Akt phosphorylation at Ser(473) and Thr(308) residues was significantly decreased, whereas Bax and cleaved caspase-3 levels were significantly increased in tumor tissues treated with both radiation and paclitaxel. The combined treatment was more effective than either treatment alone in regulating the studied molecules. We also found that paclitaxel, but not radiation, inhibited phosphoinositide 3-kinase (PI3K) activity. CONCLUSIONS: Our study suggested that a PTEN-PI3K-Akt-Bax signaling cascade is involved in the therapeutic effect of combined radiation/paclitaxel treatment in NSCLC without p53 expression. Our study also suggested that PTEN is an ideal target in tumors with wild-type PTEN and a lack of functional p53.

DNA damage after acute exposure of mice skin to physiological doses of UVB and UVA light.

Solar ultraviolet (UV) radiation is an important risk factor in skin carcinogenesis. This has been attributed mainly to the UVB waveband because the high-energetic photons are capable of interacting with DNA and inducing DNA damage. Recently, UVA light has also gained increasing interest in relation to DNA alteration. Although UVA photons are less energetic than UVB, they comprise a major fraction of sunlight UV radiation and penetrate deep into the skin. The study was carried out to compare the acute effects of UVA and UVB light on SKH-1 mice in relation to DNA damage and associated parameters. Mice were exposed to UVA (10 and 20 J/cm(2)) or UVB (200 and 800 mJ/cm(2)) radiation. The number of DNA single-strand breaks (SSB) in lymphocytes, amount of phosphorylated histone H2AX (gamma-H2AX) and apoptosis or DNA fragmentation (TUNEL-positive cells) in skin sections and level of gamma-H2AX, activated caspase-3 and phosphorylated p53 in skin were evaluated after 4 and 24 h. SSB analyzed by alkaline comet assay were found to be 4 and 24 h following UVB and UVA treatment, respectively. TUNEL and gamma-H2AX-positive cell were observed only in UVB exposed animals at both time intervals. The level of activated caspase-3 and phospho-p53 was increased 24 h after UVA and UVB radiation and was more apparent in UVB treated mice. The results indicate that the mechanism of DNA damage caused by acute UVA exposure includes formation of SSB (oxidative damage), but not double-strand breaks.

Cytotoxicity of gamma-ray in rat immature hippocampal neurons.

This in vitro study evaluated the detrimental effect of acute gamma (γ)-irradiation on rat immature hippocampal neurons. Rat immature hippocampal neurons (0.5 day in vitro) were irradiated with 0~4 Gy γ-rays. Cytotoxicity was analyzed using a lactate dehydrogenase release assay at 24 h after γ-irradiation. Radiation-induced cytotoxicity in immature hippocampal neurons increased in a dose-dependent manner. Pre-treatments of pro-apoptotic caspase inhibitors and anti-oxidative substances significantly blocked γ-irradiation-induced cytotoxicity in immature hippocampal neurons. The results suggest that the caspase-dependent cytotoxicity of γ-rays in immature hippocampal cultured neurons may be caused by oxidative stress.

[Ways of realizing apoptosis of human lymphocytes induced by UV-light and reactive oxygen species].

Changes of DNA structural condition, the level of membrane Fas-receptor expression, caspase-3 functional activity, concentrations of Ca2+, p53 and cytochrome c proteins of human lymphocytes in dynamics of apoptosis development induced by UV-light (240-390 nm) at doses 151, 1510, 3020 J/m2 and reactive oxygen species (superoxide anion-radical, hydroxyl radicals, hydrogen peroxide, singlet oxygen) have been studied. UV-light and reactive oxygen species have been established to induce fragmentation of lymphocyte DNA after 20 h incubation of the modified cells. It has been shown, that the increase in the expression level of membrane death Fas-receptors is observed during 1-5 h after exposure oflymphocytes to UV-light and ROS compared with intact cells. Also revealed is augmentation of lymphocyte caspase-3 functional activity 4 h after generation of singlet oxygen, hydroxyl radical and hydrogen peroxide addition, as well as 8 and 24 and 6 and 8 h after UV-irradiation of the cells at doses 151 and 1510 J/m2, correspondingly. Using DNA-comet method made it possible to tape that DNA damages (single-strand breaks) appear 15-20 min after lymphocyte UV-irradiation at doses 1510 and 3020 J/m and addition of hydrogen peroxide in concentration 10(-6) mol/l (C1 type comet) and reach their maximum 6 h after modification of the cells (C2 and C3 type comets). It has been observed, that 6 h after exposure oflymphocytes to hydrogen peroxide and UV-light at doses 1510 and 3020 J/m2, the p53 level of investigated cells raises. It has also been shown that the higher level of calcium in lymphocyte cytosol in conditions of UV-light exposure (1510 J/m2) and exogenous generation of reactive oxygen species is caused by Ca2+ exit from intracellular depots as a result of activating the components of the phosphoinositide mechanism for transferring information into a cell. Ideas about correlation between alterations of the calcium level and initiation of programmed cellular destruction of human lymphocytes after exposure to UV-irradiation and ROS is proposed. The authors come to the conclusion about the leading role of receptor-mediated (Fas-dependent) caspase- and p53-dependent ways of realizing apoptosis oflymphocytes induced by UV-light at doses 151 and 1510 J/m2 and active oxygen metabolites. The pattern of the possible intracellular events leading to apoptotic destruction of lymphocytes after their UV-irradiation is offered.

Mobile phone radiation does not induce pro-apoptosis effects in human spermatozoa.

Abstract Recent reports suggest that mobile phone radiation may diminish male fertility. However, the effects of this radiation on human spermatozoa are largely unknown. The present study examined effects of the radiation on induction of apoptosis-related properties in human spermatozoa. Ejaculated, density-purified, highly motile human spermatozoa were exposed to mobile phone radiation at specific absorption rates (SARs) of 2.0 and 5.7 W/kg. At various times after exposure, flow cytometry was used to examine caspase 3 activity, externalization of phosphatidylserine (PS), induction of DNA strand breaks, and generation of reactive oxygen species. Mobile phone radiation had no statistically significant effect on any of the parameters studied. This suggests that the impairment of fertility reported in some studies was not caused by the induction of apoptosis in spermatozoa.

Protective effects of tetrahydropalmatine against gamma-radiation induced damage to human endothelial cells.

AIMS: Irradiation-induced damage to pulmonary endothelial cells is thought to be an important mediator of the pathogenesis of radiation pneumonopathy. Tetrahydropalmatine (THP) has been shown to have a protective effect against oxidative stress. This study was designed to investigate the potential radioprotective effect of THP against irradiation-induced endothelial cellular damage and to elucidate the underlying mechanisms. MAIN METHODS: Human EA.hy926 cells were treated with THP and irradiation. Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For the detection of apoptosis, morphological observation, flow cytometry and a caspase-3 activity assay were employed. The expression of cytochrome-c and Bax/Bcl-2 protein were detected by western blot analysis. Generation of reactive oxygen species (ROS) was measured by flow cytometry. Malondialdehyde (MDA), lactate dehydrogenase (LDH), glutathione (GSH) and superoxide dismutase (SOD) were measured to assess cellular oxidative stress induced injury. KEY FINDINGS: Preincubation of EA.hy926 cells with THP before gamma-radiation resulted in significant inhibition of apoptosis and enhancement of cell viability, as revealed by morphological observation, flow cytometry and MTT assay. THP significantly reduced intracellular ROS formation, levels of intracellular MDA and LDH, and enhanced the production of intracellular antioxidants (GSH and SOD) in EA.hy926 cells. Meanwhile, THP also inhibited the decrease of intracellular mitochondrial membrane potential (psim), caspase-3 activation, cytochrome-c release and reduced Bax/Bcl-2 ratio in THP pretreated, irradiated cells. SIGNIFICANCE: Our findings demonstrated THP could effectively protect endothelial cells against gamma-irradiation injury, which could potentially be applied to the prevention of endothelial cell dysfunctions associated with ionizing irradiation-induced lung injury.

Gamma-ray susceptibility of immature and mature hippocampal cultured cells.

Ionizing radiation suppresses neurogenesis in the mammalian brain. This in vitro study compared the detrimental effect of acute gamma-irradiation on immature hippocampal cells with mature cells. Both rat immature (0.5 day in vitro (DIV)) and mature hippocampal cells (14 DIV) were irradiated with 0-4 Gy gamma-rays. Cell viability was analyzed by using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA fragmentation study was performed by extracting intracellular DNA. Morphological features of apoptosis were characterized by 4',6-diamidine-2'-phenylindole, dihydrochloride (DAPI) staining. MTT assay revealed that the survival rate of immature hippocampal cells declined in a dose-dependent manner within the range of irradiation applied, but was not changed in mature cells. Intranucleosomal DNA fragmentation in a ladder like pattern was dose-dependently increased in immature cells, but not in mature cells. The number of apoptotic nuclei in immature cells increased significantly in a dose-dependent manner within the range of irradiation applied. Active caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) expressions in immature hippocampal cells at 6 hr after 2 Gy exposure were markedly higher than control levels. The significantly greater radiosensitivity of immature hippocampal cells than that of the mature cells, indicates that the susceptibility of such hippocampal cells depends on their maturation. In addition, gamma-irradiation may induce caspase-dependent apoptosis in immature hippocampal cells.

Apoptin enhances radiation-induced cell death in poorly responding head and neck squamous cell carcinoma cells.

Treatment of head and neck cancers is still rather poor and worldwide new treatment options are sought. Sensitizing radioresistant tumours by combining irradiation with other therapeutics to induce apoptosis are widely investigated. We examined whether chicken anaemia virus-derived apoptin protein would have a beneficial effect on irradiation of radiosensitive SCC61 and radioresistant SQD9 human head and neck squamous carcinoma cell lines. In both cell lines, concurrent exposure to irradiation and apoptin resulted in analysed mitochondrial cytochrome c release and in cleavage of caspase-3, whereas irradiation alone of SQD9 cells under identical conditions did not. Moreover, in comparison with the irradiation, only the synchronized treatment of apoptin and irradiation resulted in increased cell death in especially the radioresistant SQD9 cells, as measured by means of a colony survival assay. Our data reveal that apoptin treatment represents an effective way for enhancing radiotherapy of tumours responding poorly to radiotherapy.

Effect of mobile phone exposure on apoptotic glial cells and status of oxidative stress in rat brain.

The aim of this study was to investigate the effects of mobile phone exposure on glial cells in brain. The study carried out on 31 Wistar Albino adult male rats. The rat heads in a carousel exposed to 900 MHz microwave. For the study group (n:14), rats exposed to the radiation 2 h per day (7 days in a week) for 10 months. For the sham group (n:7), rats were placed into the carousel and the same procedure was applied except that the generator was turned off. For the cage control (n:10), nothing applied to rats in this group. In this study, rats were euthanized after 10 months of exposure periods and brains were removed. Brain tissues were immunohistochemically stained for the active (cleaved) caspase-3, which is a well-known apoptosis marker, and p53. The expression of the proteins was evaluated by a semi-quantitative scoring system. However, total antioxidative capacity (TAC), catalase, total oxidant status (TOS), and oxidative stress index were measured in rat brain. Final score for apoptosis in the exposed group was significantly lower than the sham (p < 0.001) and the cage control groups (p < 0.01). p53 was not significantly changed by the exposure (p > 0.05). The total antioxidant capacity and catalase in the experimental group was found higher than that in the sham group (p < 0.001, p < 0.05). In terms of the TOS and oxidative stress index, there was no statistically significant difference between exposure and sham groups (p > 0.05). In conclusion, the final score for apoptosis, total antioxidant capacity and catalase in rat brain might be altered by 900 MHz radiation produced by a generator to represent exposure of global systems for mobile communication (GSM) cellular phones.