Protective effect of paeoniflorin on irradiation-induced cell damage involved in modulation of reactive oxygen species and the mitogen-activated protein kinases

Ionizing radiation can induce DNA damage and cell death by generating reactive oxygen species (ROS). The objective of this study was to investigate the radioprotective effect of paeoniflorin (PF, a main bioactive component in the traditional Chinese herb peony) on irradiated thymocytes and discover the possible mechanisms of protection. We found 60Co gamma-ray irradiation increased cell death and DNA fragmentation in a dose-dependent manner while increasing intracellular ROS. Pretreatment of thymocytes with PF (50-200 microg/ml) reversed this tendency and attenuated irradiation-induced ROS generation. Hydroxyl-scavenging action of PF in vitro was detected through electron spin resonance assay. Several anti-apoptotic characteristics of PF, including the ability to diminish cytosolic Ca2+ concentration, inhibit caspase-3 activation, and upregulate Bcl-2 and downregulate Bax in 4Gy-irradiated thymocytes were determined. Extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 kinase were activated by 4Gy irradiation, whereas its activations were partly blocked by pretreatment of cells with PF. The presence of ERK inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580 decreased cell death in 4Gy-irradiated thymocytes. These results suggest PF protects thymocytes against irradiation-induced cell damage by scavenging ROS and attenuating the activation of the mitogen-activated protein kinases.     

黄连素预处理对6-羟基多巴胺所致PC12细胞损伤的影响

目的:观察黄连素(Berberine,BBR)预处理对6-羟基多巴胺(6-hydroxydopamine,6-OHDA)诱导的PC12细胞的影响,并探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了BBR的保护作用。方法:将PC12细胞分为5组,分别为正常培养的对照组(Control)、25μM的6-OHDA损伤组、1μM的BBR预处理24 h组(BBR+6-OHDA)、SOD2-siRNA干扰组(SOD2-siRNA+BBR+6-OHDA)和乱序siRNA处理组(SC-siRNA+BBR+6-OHDA),孵育24 h后,采用噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,试剂盒检测培养基乳酸脱氢酶(Lactic Dehydrogenase,LDH)、细胞内活性氧(Reactive Oxygen Species,ROS)、还原型谷胱甘肽(Glutathione,GSH)和过氧化氢酶(Catalase,CAT)的含量,使用流式细胞仪检测凋亡率,Western blot检测SOD2和凋亡蛋白Cleaved caspase-3的表达。结果:与Control组相比,6-OHDA诱导PC12细胞24 h后,细胞活力显著降低,SOD2表达、GSH和CAT的含量明显减少,培养基上清液LDH活力、细胞凋亡率、Cleaved caspase-3表达和ROS水平显著增加(P<0.05),而BBR预处理可显著恢复6-OHDA诱导的PC12细胞活力、SOD2表达、GSH和CAT水平,并降低细胞凋亡率、凋亡蛋白表达和细胞ROS水平(P<0.05),而SOD2-siRNA显著逆转了BBR预处理产生的上述保护作用(P<0.05),SC-siRNA则未对BBR预处理产生的上述作用造成明显影响(P>0.05)。结论:黄连素预处理可减轻6-OHDA诱导的PC12细胞损伤,而SOD2分子介导了BBR预处理对暴露于6-OHDA的PC12细胞的保护作用。     

Protein oxidative damage and redox imbalance induced by ionising radiation in CHO cells

Reactive oxygen species (ROS) are important mediators of the cytotoxicity induced by the direct reaction of ionising radiation (IR) with all critical cellular components, such as proteins, lipids, and nucleic acids. The derived oxidative damage may propagate in exposed tissues in a dose- and spatiotemporal dependent manner to other cell compartments, affecting intracellular signalling, and cell fate. To understand how cell damage is induced, we studied the oxidative events occurring immediately after cell irradiation by analysing the fate of IR-derived ROS, the intracellular oxidative damage, and the modification of redox environment accumulating in Chinese hamster ovary (CHO) within 1?h after cell irradiation (dose range 0–10?Gy). By using the immuno-spin trapping technique (IST), spectrophotometric methods, and electron paramagnetic resonance (EPR) spectroscopy, we showed that IR-derived ROS (i) induced an IST-detectable, antioxidant-inhibitable one-electron oxidation of specific intracellular proteins; (ii) altered the glutathione (GSH) content (which was found to increase below 2?Gy, and decrease at higher doses, leading to a redox imbalance); (iii) decreased glutathione peroxidase and glutaredoxin activity; (iv) modified neither glutathione reductase nor thioredoxin reductase activity; (v) were detected by spin trapping technique, but adduct intensity decreased due to cell competition for ROS; and (vi) induced no EPR-detectable radicals assignable to oxidised cellular components. In conclusion, our results showed that IR generated an early high oxidising potential (protein radical intermediates, redox imbalance, modified redox enzyme activity) in irradiated cells potentially able to propagate the damage and induce oxidative modification of secondary targets.     

Photodynamic treatment with anionic nanoclays containing curcumin on human triple-negative breast cancer cells: Cellular and biochemical studies

Photodynamic treatment is a minimally invasive and clinically approved procedure for eliminating selected malignant cells with activation of a photosensitizer agent at a specific light. Little is known, however, about the phototoxic properties of curcumin, as a natural phenolic compound, against different types of cancers. It is generally accepted that cellular damage occurs during photo treatment. There is a limitation in using of curcumin as a drug due to its low solubility, but nanoparticles such as anionic nanoclays or layered double hydroxide (LDH) could overcome it. The aim of this study was to investigate cellular responses to curcumin-LDH nanoparticles after photodynamic treatment of MDA-MB-231 human breast cancer cells. For this purpose, the MDA-MB-231 human breast cancer cell line treated with curcumin-LDH nanoparticle and then irradiated (photodynamic treatment). After irradiation, lactate dehydrogenase assay, clonogenic cell survival, cell death mechanisms such as autophagy and apoptosis were determined. Cell cycle distribution after photodynamic therapy (PDT) and also intracellular reactive oxygen species (ROS) generation were measured. The result showed that curcumin-LDH–PDT has a cytotoxic and antiprolifrative effect on MDA-MB-231 human breast cancer cells. Curcumin-LDH–PDT induced autophagy, apoptosis, and G0/G1 cell cycle arrest in human breast cancer cell line. Intracellular ROS increased in MDA-MB-231 cancer cell line after treatment with curcumin-LDH along with irradiation. The results suggest that curcumin-LDH nanoparticle could be considered as a novel approach in the photodynamic treatment of breast cancer.     

Protective effects of catalase overexpression on UVB-induced apoptosis in normal human keratinocytes

UV-induced apoptosis in keratinocytes is a highly complex process in which various molecular pathways are involved. These include the extrinsic pathway via triggering of death receptors and the intrinsic pathway via DNA damage and reactive oxygen species (ROS) formation. In this study we investigated the effect of catalase and CuZn-superoxide dismutase (SOD) overexpression on apoptosis induced by UVB exposure at room temperature or 4 degrees C on normal human keratinocytes. Irradiation at low temperature reduced UV-induced apoptosis by 40% in normal keratinocytes independently of any change in p53 and with a decrease in caspase-8 activation. Catalase overexpression decreased apoptosis by 40% with a reduction of caspase-9 activation accompanied by a decrease in p53. Keeping cells at low temperature and catalase overexpression had additive effects. CuZn-SOD overexpression had no significant effect on UVB-induced apoptosis. UVB induced an increase in ROS levels at two distinct stages: immediately following irradiation and around 3 h after irradiation. Catalase overexpression inhibited only the late increase in ROS levels. We conclude that catalase overexpression has a protective role against UVB irradiation by preventing DNA damage mediated by the late ROS increase.     

Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells

Selective inhibition with sulphonamides of carbonic anhydrase (CA) IX reduces cell proliferation and induces apoptosis in human cancer cells. The effect on CA IX expression of seven previously synthesised sulphonamide inhibitors, with high affinity for CA IX, as well as their effect on the proliferation/apoptosis of cancer/normal cell lines was investigated. Two normal and three human cancer cell lines were used. Treatment resulted in dose- and time-dependent inhibition of the growth of various cancer cell lines. One compound showed remarkably high toxicity towards CA IX-positive HeLa cells. The mechanisms of apoptosis induction were determined with Annexin-V and AO/EB staining, cleaved caspases (caspase-3, caspase-8, caspase-9) and cleaved PARP activation, reactive oxygen species production (ROS), mitochondrial membrane potential (MMP), intracellular pH (pHi), extracellular pH (pHe), lactate level and cell cycle analysis. The autophagy induction mechanisms were also investigated. The modulation of apoptotic and autophagic genes (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin and LC3) was measured using real time PCR. The positive staining using γ-H2AX and AO/EB dye, showed increased cleaved caspase-3, caspase-8, caspase-9, increased ROS production, MMP and enhanced mRNA expression of apoptotic genes, suggesting that anticancer effects are also exerted through its apoptosis-inducing properties. Our results show that such sulphonamides might have the potential as new leads for detailed investigations against CA IX-positive cervical cancers.     

Bystander normal human fibroblasts reduce damage response in radiation targeted cancer cells through intercellular ROS level modulation

The radiation-induced bystander effect is a well-established phenomenon which results in damage in non-irradiated cells in response to signaling from irradiated cells. Since communication between irradiated and bystander cells could be reciprocal, we examined the mutual bystander response between irradiated cells and co-cultured with them non-irradiated recipients. Using a transwell culture system, irradiated human melanoma (Me45) cells were co-cultured with non-irradiated Me45 cells or normal human dermal fibroblasts (NHDF) and vice versa. The frequency of micronuclei and of apoptosis, ROS level, and mitochondrial membrane potential were used as the endpoints. Irradiated Me45 and NHDF cells induced conventional bystander effects detected as modest increases of the frequency of micronuclei and apoptosis in both recipient neighbors; the increase of apoptosis was especially high in NHDF cells co-cultured with irradiated Me45 cells. However, the frequencies of micronuclei and apoptosis in irradiated Me45 cells co-cultured with NHDF cells were significantly reduced in comparison with those cultured alone. This protective effect was not observed when irradiated melanomas were co-cultured with non-irradiated cells of the same line, or when irradiated NHDF fibroblasts were co-cultured with bystander melanomas. The increase of micronuclei and apoptosis in irradiated Me45 cells was paralleled by an increase in the level of intracellular reactive oxygen species (ROS), which was reduced significantly when they were co-cultured for 24h with NHDF cells. A small but significant elevation of ROS level in NHDF cells shortly after irradiation was also reduced by co-culture with non-irradiated NHDF cells. We propose that in response to signals from irradiated cells, non-irradiated NHDF cells trigger rescue signals, whose nature remains to be elucidated, which modify the redox status in irradiated cells. This inverse bystander effect may potentially have implications in clinical radiotherapy.     

Bystander response in human lymphoblastoid TK6 cells

The mechanisms of the medium-mediated bystander response induced by γ-rays in non-irradiated TK6 cells were investigated. Cell cultures were irradiated and the culture medium discarded immediately after irradiation and replaced with a fresh one. In cells incubated with conditioned medium from irradiated cells (CM), a significant decrease in cell viability and cloning efficiency was observed, together with a significant increase in apoptosis, also in directly irradiated cells. To examine whether bystander apoptosis involved the extrinsic pathway, an inhibitor of caspase-8 was added to CM cultures, which significantly decreased apoptosis to control levels. The addition to CM of ROS scavengers, Cu–Zn superoxide dismutase and N-acetylcysteine did not affect the induction of apoptosis. To assess whether CM treatment activates a DNA damage response, also the formation of γ-H2AX foci, as markers of double-strand breaks and their colocalisation with 53-binding protein 1 (53BP1) and the protein mutated in the Nijmegen breakage syndrome 1 (NBS1) was analysed. In cultures treated for 2 h with CM, 9–11% of cells showed γ-H2AX foci, which partially or totally lacked colocalisation with 53BP1 and NBS1 foci. About 85% of irradiated cells were positive for γ-H2AX foci, which colocalised with 53BP1 and NBS1 proteins. At 24 h from irradiation, very few irradiated cells retained foci, fitting DNA repair kinetics. The number of foci-positive bystander cells also decreased to background values 24 h after CM incubation. Our results suggest that irradiated TK6 cells release into the medium some soluble factors, not ROS, which are responsible for the cytotoxic effects induced in bystander cells. In our experimental system, the role of ROS appeared to be of minor importance in inducing cell mortality, but probably critical in activating the DNA damage response in the responsive fraction of bystander cells.     

Hyperoside prevents oxidative damage induced by hydrogen peroxide in lung fibroblast cells via an antioxidant effect

We elucidated the cytoprotective effects of hyperoside (quercetin-3-O-galactoside) against hydrogen peroxide (H2O2)-induced cell damage. We found that hyperoside scavenged the intracellular reactive oxygen species (ROS) detected by fluorescence spectrometry, flow cytometry, and confocal microscopy. In addition, we found that hyperoside scavenged the hydroxyl radicals generated by the Fenton reaction (FeSO4)+H2O2) in a cell-free system, which was detected by electron spin resonance (ESR) spectrometry. Hyperoside was found to inhibit H2O2-induced apoptosis in Chinese hamster lung fibroblast (V79-4) cells, as shown by decreased apoptotic nuclear fragmentation, decreased sub-G(1) cell population, and decreased DNA fragmentation. In addition, hyperoside pretreatment inhibited the H2O2-induced activation of caspase-3 measured in terms of levels of cleaved caspase-3. Hyperoside prevented H2O2-induced lipid peroxidation as well as protein carbonyl. In addition, hyperoside prevented the H2O2-induced cellular DNA damage, which was established by comet tail, and phospho histone H2A.X expression. Furthermore, hyperoside increased the catalase and glutathione peroxidase activities. Conversely, the catalase inhibitor abolished the cytoprotective effect of hyperoside from H2O2-induced cell damage. In conclusion, hyperoside was shown to possess cytoprotective properties against oxidative stress by scavenging intracellular ROS and enhancing antioxidant enzyme activity.     

Inhibition of UVA-induced apoptotic signaling pathway by polypeptide from <Emphasis Type="Italic">Chlamys farreri</Emphasis> in human HaCaT keratinocytes

Chronic UVA irradiation has been reported to induce photoaging and photocarcinogenesis. UVA is a potent inducer of reactive oxygen species (ROS), which can induce various biological processes, including apoptosis. Polypeptide from Chlamys farreri (PCF) is a novel marine active material isolated from the gonochoric Chinese scallop C. farreri. In our previous studies, PCF was found to be an effective antioxidant inhibiting UVA-induced ROS production and a potential inhibitory agent for UVA-induced apoptosis in the human keratinocyte cell line HaCaT. The intracellular mechanisms of how PCF protects HaCaT cells from UVA-induced apoptosis are not understood. Thus, we here investigate the effect of PCF on UVA-induced intracellular signaling of apoptosis. Pretreatment with the ROS scavenger N-acetylcysteine (NAC), the p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor Ac-DEVD-CHO was found to effectively prevent UVA-induced apoptosis, indicating that ROS, p38 MAPK and caspase-3 play important roles in apoptosis. H₂O₂-induced apoptosis was attenuated by PCF, suggesting that PCF plays its anti-apoptotic role through its antioxidant activity. In addition, PCF treatment inhibited UVA-induced p38 MAPK activation and caspase-3 activation, as assayed by Western blot analysis and flow cytometry, respectively. Our results suggest that PCF attenuates UVA-induced apoptosis through a reduction of ROS generation and diminished p38 MAPK and caspase-3 activation.     

Polypeptide from Chlamys farreri inhibits UVB-induced HaCaT cells apoptosis via inhibition CD95 pathway and reactive oxygen species

Polypeptide from Chlamys farreri (PCF) is a novel marine active product isolated from gonochoric Chinese scallop Chlamys farreri which has recently been found to be an effective antioxidant. In this study, we assessed the effect of PCF on UVB-induced intracellular signalling of apoptosis in HaCaT cells. Pre-treatment with PCF significantly inhibited UVB-induced apoptosis in HaCaT cells. PCF strongly reduced the intracellular reactive oxygen species (ROS) level followed by inhibiting the release of cytochrome c. The expression of CD95 and Fas-associating protein with death domain (FADD) was eliminated in a dose-dependent manner by PCF pre-treatment in UVB-irradiated HaCaT cells, followed by inhibition of cleavage of procaspase-8, whose activation induced cell apoptosis. Furthermore, pre-treatment with the ROS scavenger N-acetylcysteine (NAC) and the caspase-8 inhibitor z-IETD-fmk was found to effectively prevent UVB-induced apoptosis, suggesting that UVB-induced HaCaT cell apoptosis was partially due to generation of ROS and activation of the caspase-8 pathway. Consequently, the protective effect of PCF against UVB irradiation in HaCaT cells is exerted by suppression of generation of ROS followed by inhibition of cytochrome c release and inactivation of Fas-FADD-caspase-8 pathway, resulting in blockage of UVB-induced apoptosis.     

Effect of polyamines on superoxide dismutase activity under dexamethasone-induced apoptosis in rat thymocytes

The intracellular redox state is of importance for cell growth, differentiation, and apoptosis through reactive oxygen species (ROS) functioning as metabolic fine-tuner. Optimal levels of polyamines are necessary for growth, differentiation, and apoptotic cell death while they also protect cell from ROS accumulation. We have carried out studies to find out the interrelation between these two distant metabolic pathways. For that purpose, the glucocorticoid-triggered programmed cell death of rat thymocytes has been used. Our data confirm that SOD activity (which testifies both to the level of ROS generation and antioxidative defense state) changes in response to programmed cell death conditions and to alteration of intracellular polyamines level. Thymocytes death induced by dexamethasone is partially mediated by polyamines content. Our data prove that one of the molecular mechanisms of thymocytes population resistance after dexamethasone treatment is an enhanced level of antioxidant defense. It is evident that in dexamethasone-treated rat thymocytes polyamines modulate signal transduction processes to apoptosis development via changes in cellular redox status.     

Lead‐induced DNA damage and cell apoptosis in human renal proximal tubular epithelial cell: Attenuation via N‐acetyl cysteine and tannic acid

This study investigates the exposure of lead‐induced reactive oxygen species (ROS) generation, DNA damage, and apoptosis and also evaluates the therapeutic intervention using antioxidants in human renal proximal tubular cells (HK‐2 cells). Following treatment of HK‐2 cells with an increasing concentration of lead nitrate (0–50 μM) for 24 h, the intracellular ROS level increased whereas the GSH level decreased significantly in a dose‐dependent manner. Comet assay results revealed that lead nitrate showed the ability to increase the levels of DNA strand breaks in HK‐2 cells. Lead exposure also induced apoptosis through caspase‐3 activation at 30 μg/mL. Pretreatment with N‐acetylcysteine (NAC) and tannic acid showed a significant ameliorating effect on lead‐induced ROS, DNA damage, and apoptosis. In conclusion, lead induces ROS, which may exacerbate the DNA damage and apoptosis via caspase‐3 activation. Additionally, supplementation of antioxidants such as NAC and tannic acid may be used as salvage therapy for lead‐induced DNA damage and apoptosis in an exposed person.     

The mechanisms of oxidative DNA damage and apoptosis induced by norsalsolinol, an endogenous tetrahydroisoquinoline derivative associated with Parkinson's disease

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N -acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using ³²P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H₂O₂ with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.     

Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells: role of oxidant-induced iron signaling in apoptosis

In the past, investigators have successfully used iron chelators to mitigate the cardiotoxicity of doxorubicin (DOX), a widely used anticancer drug that induces reactive oxygen species (ROS), oxidative damage, and apoptosis. Although intracellular iron plays a critical role in initiating DOX-induced apoptosis, the molecular mechanism(s) that link iron, ROS, and apoptosis are still unknown. In this study, we demonstrate that apoptosis results from the exposure of bovine aortic endothelial cells to DOX and that the apoptotic cell death is accompanied by a significant increase in cellular iron ((55)Fe) uptake and activation of iron regulatory protein-1. Furthermore, DOX-induced iron uptake was shown to be mediated by the transferrin receptor (TfR)-dependent mechanism. Treatment with the anti-TfR antibody (IgA class) dramatically inhibited DOX-induced apoptosis, iron uptake, and intracellular oxidant formation as measured by fluorescence using dichlorodihydrofluorescein. Treatment with cell-permeable iron chelators and ROS scavengers inhibited DOX-induced cellular (55)Fe uptake, ROS formation, and apoptosis. Based on these findings, we conclude that DOX-induced iron signaling is regulated by the cell surface TfR expression, intracellular oxidant levels, and iron regulatory proteins. The implications of TfR-dependent iron transport in oxidant-induced apoptosis in endothelial cells are discussed.     

Role of ceramide in mediating apoptosis of irradiated LNCaP prostate cancer cells

The sphingomyelin metabolites ceramide and sphingosine are mediators of cell death induced by gamma-irradiation. We studied the production of ceramide and the effects of exogenous ceramide on apoptosis in LNCaP prostate cancer cells that are highly resistant to gamma-irradiation-induced cell death. LNCaP cells can be sensitized to gamma-irradiation by tumor necrosis factor alpha (TNF-alpha) and, to a lesser degree, by the agonistic FAS antibody CH-11. TNF-alpha activated intrinsic and extrinsic apoptosis pathways and increased ceramide and sphingosine levels in irradiated LNCaP cells. CH-11 activated only the extrinsic apoptosis pathways and had a negligible effect on ceramide and sphingosine levels in irradiated LNCaP cells. Exogenous ceramide and bacterial sphingomyelinase sensitized LNCaP cells to radiation-induced apoptosis and had a synergistic effect on cell death after irradiation with TNF-alpha, but not with CH-11. Cell death effects after exposure to ceramide and irradiation were blocked by the serine protease inhibitor TLCK (Na-p-tosyl-L-lysine-chloromethylketone), but not by the caspase inhibitor z-VAD (2-val-Ala-Asp(oMe)-CH(2)F). During LNCaP cell apoptosis induced by exogenous ceramide, we observed activation of caspase-9, but not caspases-8, -3, or -7. The effect of ceramide occurred largely via the intrinsic mitochondrial apoptosis pathway and enhanced TNF-alpha, but not CH-11 effects on irradiated cells. The data show that ceramide enhanced activation of the intrinsic apoptotic pathway and enhanced cell death induced by TNF-alpha with or without gamma-irradiation. TNF-alpha and gamma-irradiation elevated levels of endogenous ceramide and activated the intrinsic cell death pathway.     

Polypeptide from Chlamys farreri inhibits UVB-induced HaCaT cells apoptosis via inhibition CD95 pathway and reactive oxygen species

Polypeptide from Chlamys farreri (PCF) is a novel marine active product isolated from gonochoric Chinese scallop Chlamys farreri which has recently been found to be an effective antioxidant. In this study, we assessed the effect of PCF on UVB-induced intracellular signalling of apoptosis in HaCaT cells. Pre-treatment with PCF significantly inhibited UVB-induced apoptosis in HaCaT cells. PCF strongly reduced the intracellular reactive oxygen species (ROS) level followed by inhibiting the release of cytochrome c. The expression of CD95 and Fas-associating protein with death domain (FADD) was eliminated in a dose-dependent manner by PCF pre-treatment in UVB-irradiated HaCaT cells, followed by inhibition of cleavage of procaspase-8, whose activation induced cell apoptosis. Furthermore, pre-treatment with the ROS scavenger N-acetylcysteine (NAC) and the caspase-8 inhibitor z-IETD-fmk was found to effectively prevent UVB-induced apoptosis, suggesting that UVB-induced HaCaT cell apoptosis was partially due to generation of ROS and activation of the caspase-8 pathway. Consequently, the protective effect of PCF against UVB irradiation in HaCaT cells is exerted by suppression of generation of ROS followed by inhibition of cytochrome c release and inactivation of Fas-FADD–caspase-8 pathway, resulting in blockage of UVB-induced apoptosis.     

Cytoprotective activity of annphenone against oxidative stress-induced apoptosis in V79-4 lung fibroblast cells

We have elucidated the cytoprotective effect of annphenone (2,4-dihyroxy-6-methoxy-acetophenone 4-O-beta-d-glucopyranoside) against oxidative stress-induced apoptosis. Annphenone scavenged intracellular reactive oxygen species (ROS) and increased antioxidant enzyme activities. It thereby prevented lipid peroxidation and DNA damage, which was demonstrated by the inhibition of the formation of thiobarbituric acid reactive substance (TBARS), inhibition of the comet tail and decreased phospho-H2A.X expression. Annphenone protected Chinese hamster lung fibroblast (V79-4) cells from cell death via the inhibition of apoptosis induced by hydrogen peroxide (H(2)O(2)), as shown by decreased apoptotic nuclear fragmentation, decreased sub-G(1) cell population and inhibited mitochondrial membrane potential (Deltapsi) loss. Taken together, these findings suggest that annphenone exhibits antioxidant properties by inhibiting ROS generation and thus protecting cells from H(2)O(2)-induced cell damage.     

Reactive oxygen species‐induced parthanatos of immunocytes by human cytomegalovirus‐associated substance

Previous studies have examined various immune evasion strategies of human cytomegalovirus (HCMV) to gain understanding of its pathogenesis. Although the mechanism that underlies immunocyte destruction near HCMV‐infected lesions has yet to be established, it is here shown that substances produced by HCMV‐infected cells induce death in several types of immunocytes, but not in fibroblasts or astrocytomas. These substances contain HCMV proteins and were termed HCMV‐associated insoluble substance (HCMVAIS). The mechanism by which HCMVAIS induces cell death was characterized to improve understanding the death of immunocytes near HCMV‐infected lesions. HCMVAIS were found to trigger production of intracellular nicotinamide adenine dinucleotide phosphate oxidase‐derived reactive oxygen species (ROS), resulting in cell death, this effect being reversed following treatment with ROS inhibitors. Cell death was not induced in splenocytes from NOX‐2 knockout mice. It was hypothesized that DNA damage induced by oxidative stress initiates poly ADP‐ribose polymerase‐1 (PARP‐1)‐mediated cell death, or parthanatos. HCMVAIS‐induced cell death is accompanied by PARP‐1 activation in a caspase‐independent manner, nuclear translocation of apoptosis‐inducing factor (AIF), and DNA fragmentation, which are typical features of parthanatos. Treatment with an AIF inhibitor decreased the rate of HCMVAIS‐induced cell death, this being confirmed by hematoxylin and eosin staining; cell death in most HCMV‐positive foci in serial section samples of a large intestine with HCMV infection was TUNEL‐positive, cleaved caspase 3‐negative and CD45‐positive. Taken together, these data suggest that HCMV inhibits local immune responses via direct killing of immunocytes near HCMV‐infected cells through ROS‐induced parthanatos by HCMVAIS.