Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts

Ultraviolet Al (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340–450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dosedependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.     

Arsenic trioxide-induced apoptosis and its enhancement by buthionine sulfoximine in hepatocellular carcinoma cell lines

We treated four hepatocellular carcinoma cell lines, HLE, HLF, HuH7, and HepG2 with ATO and demonstrated that arsenic trioxide (ATO) at low doses (1--3 muM) induced a concentration-dependent suppression of cell growth in HLE, HLF, and HuH7. HLE cells underwent apoptosis at 2 microM ATO, which was executed by the activation of caspase-3 through the mitochondrial pathway mediated by caspase-8 activation and Bid truncation. When these cell lines were exposed to ATO in combination with l-S,R-buthionine sulfoximine (BSO) which inhibits GSH synthesis, a synergistic growth suppression was induced, even in HepG2 showing a lower sensitivity to ATO than other cell lines tested. The intracellular GSH levels after the treatment with ATO plus BSO were considerably decreased in HLE cells compared with those after the treatment with ATO or BSO alone. The production of reactive oxygen species (ROS) which was examined by 2' ,7' -dichlorodihydrofluorescein diacetate, increased significantly after the treatment with ATO plus BSO in HLE cells. These findings indicate that ATO at low concentrations induces growth inhibition and apoptosis, and furthermore that the ATO-BSO combination treatment enhances apoptosis through increased production of ROS in hepatocellular carcinoma cells.     

Temperature-dependent,neurotrophic factor-elicited,neuronal differentiation in adrenal chromaffin cell line immortalized with temperature-sensitive SV40 T-antigen

We established adrenal medullary cell lines from transgenic mice expressing an oncogene, the temperature-sensitive simian virus 40 large T-antigen, under the control of the tyrosine hydroxylase promoter. A clonal cell line, named tsAM5D, conditionally grew at a permissive temperature of 33 degrees C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine-synthesizing enzymes and secretory vesicle-associated proteins. tsAM5D cells proliferated at the permissive temperature in response to basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). At a non-permissive temperature of 39 degrees C, bFGF and CNTF acted synergistically to differentiate tsAM5D cells into neuron-like cells. In addition, tsAM5D cells caused to differentiate by bFGF plus CNTF at 39 degrees C became dependent solely on nerve growth factor for their survival and showed markedly enhanced neurite outgrowth. In the presence of bFGF and CNTF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal marker genes including neuron-specific enolase, growth-associated protein-43, microtubule-associated protein 2, neurofilament, and p75 neurotrophin receptor, indicating that the cells underwent neuronal differentiation. Thus, we demonstrated that tsAM5D cells could proliferate at permissive 33 degrees C, and also had the capacity to terminally differentiate into neuron-like cells in response to bFGF and CNTF when the oncogene was inactivated by shifting the temperature to non-permissive 39 degrees C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation.     

Calmodulin-dependent and -independent apoptosis in cells of a Drosophila neuronal cell line

This study was undertaken to reveal apoptotic pathways in neurons using a Drosophila neuronal cell line derived from larval central nervous system. We could induce apoptotic cell death in the cells by a Ca²⁺ ionophore (A23187), a protein kinase inhibitor (H-7), an RNA synthesis inhibitor (actinomycin D) and a protein synthesis inhibitor (cycloheximide). All the apoptosis induced by each chemical required Ca²⁺ ions, although the origin of Ca²⁺ ions were different: apoptosis induced by A23187 was dependent on extracellular Ca²⁺ ions whereas those by the other three chemicals utilized intracellular Ca²⁺ ions. Furthermore, different reactions to W-7, a calmodulin inhibitor, were found: W-7 prevented the cell death by each of the three chemicals but not by A23187. Based on the results, we proposed that the apoptotic pathways are classified into two types in individual cells. One pathway induced by H-7, actinomycin D or cycloheximide is calmodulin-dependent (pathway H), and another induced by A23187 is calmodulin-independent (pathway A).     

Deacetyl epoxyazadiradione protects aminoglycoside antibiotic-induced renal cell apoptosis,in vitro

Aminoglycoside antibiotics such as gentamicin are used frequently to treat bacterial infections in humans. Excessive consumption of these antibiotics lead to renal dysfunction. One of the factors contributing to renal dysfunction is oxidative damage, which causes apoptosis. Hence, this study investigates the effect of the antioxidant compound deacetyl epoxyazadiradione (DEA) in reducing cell death induced by gentamicin treatment in kidney cells (Madin–Darby canine kidney cells). The antioxidant experiments showed that reactive oxygen species level is decreased up to 27.06 ± 0.18% in 150 µM of DEA treatment. At this concentration, the activity of antioxidant enzymes such as superoxide dismutase increased from 0.4 ± 0.04 to 1.46 ± 0.05 µmol/min/L and catalase increased from 7.48 ± 0.39 to 17.6 ± 0.74 U/mg. The relative folds of gene expression of mitochondrial enzymes such as GST, GPx and GR restored from 0.596 ± 0.019, 0.521 ± 0.013 and 0.775 ± 0.014 to 0.866 ± 0.013, 0.669 ± 0.015 and 0.8615 ± 0.028, respectively. Consequently, the percentage of cell viability increases upto 91.8 ± 2.01 from 61.93 ± 1.63 with much less fragmentation in genomic DNA. Additionally, molecular docking results showed that DEA could bind to Bax, Bcl- 2, Caspase- 3 and Caspase- 9 proteins. These results indicate that DEA could reduce cell apoptosis by reducing oxidative stress due to antibiotics and interrupting the apoptotic signal pathway in kidney cells.     

Lycium barbarum polysaccharide protects against LPS-induced ARDS by inhibiting apoptosis,oxidative stress,and inflammation in pulmonary endothelial cells

Acute respiratory distress syndrome (ARDS) is a heterogenous syndrome characterised by diffuse alveolar damage, with an increase in lung endothelial and epithelial permeability. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses antiapoptotic and antioxidative effects in distinct situations. In the present study, the protective effects and potential molecular mechanisms of LBP against lipopolysaccharide (LPS)-induced ARDS were investigated in the mice and in the human pulmonary microvascular endothelial cells (HPMECs). The data indicated that pretreatment with LBP significantly attenuated LPS-induced lung inflammation and pulmonary oedema in vivo. LBP significantly reversed LPS-induced decrease in cell viability, increase in apoptosis and oxidative stress via inhibiting caspase-3 activation and intracellular reactive oxygen species (ROS) production in vitro. Moreover, the scratch assay verified that LBP restored the dysfunction of endothelial cells (ECs) migration induced by LPS stimulation. Furthermore, LBP also significantly suppressed LPS-induced NF-κB activation, and subsequently reversed the release of cytochrome c. These results showed the antiapoptosis and antioxidant LBP could partially protect against LPS-induced ARDS through promoting the ECs survival and scavenging ROS via inhibition of NF-κB signalling pathway. Thus, LBP could be potentially used for ARDS against pulmonary inflammation and pulmonary oedema.     

Palmitoyl protein thioesterase 1 protects against apoptosis mediated by Ras-Akt-caspase pathway in neuroblastoma cells

Palmitoyl protein thioesterase (PPT) 1 is an enzyme involved in deacylation of palmitoylated proteins. A deficiency in PPT1 results in a genetic disease, infantile neuronal ceroid lipofuscinosis, associated with massive death of cortical neurons. The role of PPT1 in neuronal survival and apoptosis was studied in human neuroblastoma (LA-N-5) cells overexpressing PPT1. Overexpression of PPT1 was shown both by the 200-350% increase in depalmitoylating activity over basal level (as determined by an in vitro PPT assay) and by western blot analysis of transiently expressed epitope-tagged PPT1. Overexpressed PPT1 showed the same acidic pH optimum (pH 4.0) as the endogenous enzyme, when assayed with a P0-derived octapeptide substrate, and reduced the growth rate by 30%. LA-N-5 cells underwent apoptosis, as evidenced by increased caspase 3-like activity and increased DNA fragmentation, when challenged with either C2-ceramide or a phosphatidylinositol 3-kinase inhibitor (LY294002). Overexpression of PPT1 inhibited this C2-ceramide- or LY294002-mediated activation of caspase-3 by 50%. There was also a concomitant decrease in DNA fragmentation and cell death. Consistent with increased resistance to apoptosis, we found increased phosphorylation of the antiapoptotic protein Akt (protein kinase B) in PPT1-overexpressing cells. p21Ras is known to be dynamically palmitoylated and depalmitoylated and is involved in both growth and cell death. The C2-ceramide-induced membrane association of p21Ras was reduced by 30-50% in PPT1-overexpressing cells compared with control. PPT overexpression also led to reduced membrane association of another palmitoylated protein, GAP-43, a neuron-specific protein. Our studies suggest that protein palmitoylation could be a physiological regulator of apoptosis.     

Phase II enzyme induction by a carotenoid,lutein, in a PC12D neuronal cell line

The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels, implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.     

Establishment and characterization of a new murine mammary tumor cell line,balb/c-MC

Summary A new murine mammary tumor cell line (BALB/c-MC) was established from a spontaneous mammary tumor in a 17-mo.-old female mouse of the low mammary cancer strain BALB/cHe. The cell line was derived from a papillary adenocarcinoma. In monolayer culture the line exhibits a pavementlike arrangement of cells and forms “domes” or “hemicysts” as the cells become confluent. The cell line rapidly forms tumors when transplanted into young syngeneic BALB/cHe mice. The subcutaneous injection of 10⁶ cells resulted in the development of mammary tumors (typical papillary adenocarcinomas) in 33 of 37 (87% recipients within 2 to 3 mo. after injection. These mammary tumors also metastasize to lung [14 of 33 (42%) of recipients] during this time. The number of chromosomes in this cell line is hyperdiploid (average of 43, range 39 to 44).     

Inhibition of bioenergetics alters intracellular calcium,membrane composition,and fluidity in a neuronal cell line

The effect of inhibited bioenergetics and ATP depletion on membrane composition and fluidity was examined in cultured neuroblastoma-glioma hybrid NG108-15 cells. Sodium cyanide (CN) and 2-deoxyglucose (2-DG) were used to block oxidative phosphorylation and anaerobic glycolysis, respectively. Endoplasmic reticulum (ER) Ca²⁺-pump activity measured by⁴⁵Ca²⁺ uptake was >92% inhibited in intact cells incubated with CN (1 mM) and 2-DG (20 mM) for 30 min. In addition, exposure of cells to CN and 2-DG caused a 134% increased release of isotopically labeled arachidonic acid (³H-AA) or arachidonate-derived metabolites from membranes. Removal of Ca²⁺ from the incubation medium ablated the CN/2-DG induced release of³H-AA or its metabolites. Membrane fluidity of intact cells was measured by electron spin resonance spectroscopy using the spin label 12-doxyl stearic acid. The mean rotational correlation time (_c) of the spin label increased 49% in CN/2-DG exposed cells compared to controls, indicating a decrease in membrane fluidity. These results show that depletion of cellular ATP results in inhibition of the ER Ca²⁺-pump, loss of AA from membranes, and decreased membrane fluidity. We propose that impaired bioenergetics can increase intracellular Ca²⁺ as a result of Ca²⁺-pump inhibition and thereby activate Ca²⁺-dependent phospholipases causing membrane effects. Since neurons derive energy predominantly from oxidative metabolism, ATP depletion during brain hypoxia may initiate a similar cytotoxic mechanism.     

Antisense EGFR sequence enhances apoptosis in a human hepatoma cell line BEL—7404

Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells.In the cells of JX-1,a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research,3:75,1993),an enhanced rate(9.5%) of spontaneous apoptosis was detected by flow cytometry,whereas the rates of spontaneous apoptosis in JX-0 cells,a sub-clone of BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 cells were almost equal and about 1.7%.Serum-starvation for 72h increased the rate of apoptosis of JX-lcells up to 33.7%,while JX-0 and BEL-7404 cells,under the same condition,produced less than 5% of apoptotic cells.Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies often occurred in JX-1 cells,especially during serumstarvation.These results,combined with the data of DNA fragmentation Elisa test,suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca^2 level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0 cells indicated that antisense egfr might interrupt the EGF/EGFR sigaling pathway resulting in the decreass of intracellular Ca^2 pool content as well as the responsiveness of these cells to the extracellular signals.These findings suggest that antisense EGFR either directly or indirectly regulates Ca^2 storage in endoplasmic reticulum,thereby enhances apoptosis in the human hepatoma cells.     

Zinc toxicity in various lung cell lines is mediated by glutathione and GSSG reductase activity

In a previous work, it was shown that in cells after a decrease of cellular glutathione content, toxic zinc effects, such as protein synthesis inhibition or GSSG (glutathione, oxidized form) increases, were enhanced. In this study, zinc toxicity was determined by detection of methionine incorporation as a parameter of protein synthesis and GSSG increase in various lung cell lines (A549, L2, 11Lu, 16Lu), dependent on enhanced GSSG reductase activities and changed glutathione contents. After pretreatment of cells with dl-buthionine-[R,S]-sulfoximine (BSO) for 72 h, cellular glutathione contents were decreased to 15–40% and GSSG reductase activity was increased to 120–135% in a concentration-dependent manner. In BSO pretreated cells, the IC₅₀ values of zinc for methionine incorporation inhibition were unchanged as compared to cells not pretreated. The GSSG increase in BSO pretreated cells by zinc was enhanced in L2, 11Lu, and 16Lu cells, whereas in A549 cells, the GSSG increase by zinc was enhanced only after pretreatment with the highest BSO concentration. Inhibition of GSSG reductase in alveolar epithelial cells was observed at lower zinc concentrations than needed for methionine incorporation inhibition, whereas in fibroblastlike cells, inhibition of GSSG reductase occurred at markedly higher zinc concentrations as compared to methionine incorporation inhibition. These results demonstrate that GSSG reductase is an important factor in cellular zinc susceptibility. We conclude that reduction of GSSG is reduced in zinc-exposed cells. Therefore, protection of GSH oxidation by various antioxidants as well as enhancement of GSH content are expected to be mechanisms of diminishing toxic cellular effects after exposure to zinc.     

Neurite outgrowth promoting activity of marine algae from Japan against rat adrenal medulla pheochromocytoma cell line, PC12D

We screened for biological activity which induces neurite outgrowth in vitro from 300 species of marine algae from along the Japan coast for possible use as a treatment for the lack of neurotrophic factor which is considered to be a cause of Alzheimer's disease. In this study, we evaluated the neurite outgrowth promoting activity in a rat adrenal medulla pheochromocytoma cell line, PC12D, using a low level of NGF (nerve growth factor). Although most of the samples had no activity, MeOH extract from a brown alga, Sargassum macrocarpum and PBS extract from a red alga, Jania adharens, exhibited neurite outgrowth promoting activity and induced neuron specific dendrites and axons from the surfaces of PC12D cells. The active substance present in S. macrocarpumseemed to be lipid and heat stable with molecular weight of around 500 to 1000. These results suggest that marine algae may constitute a good source for development of promising novel agents with neurotrophic activity in brain nerve systems for future use in treatment of Alzheimer's disease. This revised version was published online in August 2006 with corrections to the Cover Date.     

Diacylglycerol kinase inhibitor R59022-induced autophagy and apoptosis in the neuronal cell line NG108-15

Phosphatidic acid (PA) is a lipid second messenger and is believed to be involved in cell proliferation and survival. PA is mainly produced by phospholipase D (PLD) and diacylglycerol kinase (DGK). Elevated PLD activity is believed to suppress apoptosis via activation of the mammalian target of rapamycin (mTOR). On the other hand, DGK inhibition has been demonstrated to induce apoptosis, but it is unclear whether DGK can regulate mTOR. Here, we investigated whether DGK inhibition can induce apoptosis and autophagy in neuronal cells, since mTOR is a key mediator of autophagy and the simultaneous activation of apoptosis and autophagy has been detected. A DGK inhibitor, R59022 induced autophagy and apoptosis without serum in NG108-15 cells. Autophagy preceded apoptosis, and apoptosis inhibition did not affect R59022-induced autophagy. R59022-induced autophagy was inhibited by exogenous PA, and protein kinase C activation and increases in intracellular Ca²⁺ levels, which are assumed to be caused by diacylglycerol accumulation, did not appear to be involved in R59022-induced autophagy. We also investigated the effects of R59022 on mTOR signaling pathway, and found that the pathway was not inhibited by R59022. These results imply that DGK plays an important role in cell survival via mTOR-independent mechanism.     

Induction of apoptosis in normal cultured rat hepatocytes and in Hep3B, a human hepatoma cell line

The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions. Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells. In this study, we compared the effects of several apoptosis-inducing agents – transforming growth factor β₁ (TGF-β₁), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) – on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h. Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining. DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot. Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry. Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA. Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-β₁; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively). OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-β₁, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h. In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis. In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Apigenin,a potent suppressor of dendritic cell maturation and migration,protects against collagen‐induced arthritis

This study aimed to investigate whether apigenin (API) suppresses arthritis development through the modulation of dendritic cell functions. Bone marrow‐derived dendritic cells (BMDCs) were stimulated in vitro with lipopolysaccharide (LPS) and treated with API for 24 hrs; DC functions, including phenotype expressions, cytokine secretion, phagocytosis and chemotaxis, were then investigated. The effects of API on collagen‐induced arthritis (CIA) were examined in vivo, and purified DCs from the lymph nodes (LNs) of API‐treated CIA mice were analysed for phenotypes and subsets. In in vitro, API efficiently restrained the phenotypic and functional maturation of LPS‐stimulated BMDCs while maintaining phagocytotic capabilities. Moreover, API inhibited the chemotactic responses of LPS‐stimulated BMDCs, which may be related to the depressive effect on chemokine receptor 4 (CXCR4). In in vivo, API treatment delayed the onset and reduced the severity of arthritis in CIA mice, and diminished secretion of pro‐inflammatory cytokines in the serum and supernatants from the LN cells of the CIA mice. Similar to the in vitro findings, the API‐treated mice exhibited reduced expression of co‐stimulatory molecules and major histocompatibility complex II on DCs. Furthermore, API treatment strongly down‐regulated the number of Langerhans cells, but not plasmacytoid DCs (pDCs) in LNs, which may be related to the depressive effect of API on the expression of CXCR4 on DCs of peripheral blood. These data provide new insight into the mechanism of action of API on arthritis and indicate that the inhibition of maturation and migration of DCs by API may contribute to its immunosuppressive effects.     

4-Phenylbutyric acid protects against neuronal cell death by primarily acting as a chemical chaperone rather than histone deacetylase inhibitor

This letter describes the mechanism behind the protective effect of 4-phenylbutyric acid (4-PBA) against endoplasmic reticulum (ER) stress-induced neuronal cell death using three simple 4-(p-substituted phenyl) butyric acids (4-PBA derivatives). Their relative human histone deacetylase (HDAC) inhibitory activities were consistent with a structural model of their binding to HDAC7, and their ability to suppress neuronal cell death and activity of chemical chaperone in vitro. These data suggest that 4-PBA protects against neuronal cell death mediated by the chemical chaperone activity rather than by inhibition of histone deacetylase.