Cd-induced Cell Death Research Articles

Luteolin prevents cadmium-induced PC12 cell death by suppressing the Akt/mTOR signaling pathway.

Cadmium (Cd) is an environmental pollutant that can cause neurodegenerative disorders. Luteolin (Lut) is a natural flavonoid compound. However, whether Lut protects against Cd-induced nerve cell death remains unclear. In the present study, PC12 cells were used to investigate the neuroprotective effect of Lut against Cd poisoning. Changes in cell viability, apoptosis, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein expression, and protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway activity were analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining, flow cytometry, and western blotting. Lut markedly attenuated the Cd-induced reduction in cell viability, nuclear fragmentation, condensation, and the decrease in the Bcl-2/Bcl-2-associated X protein ratio in PC12 cells. Furthermore, Lut blocked the Cd-mediated activation of the Akt/mTOR signaling pathway. Moreover, inhibition of the Akt/mTOR signaling pathway with LY294002 (a PI3K inhibitor) enhanced the protective effect of Lut against Cd-induced cell death by suppressing Cd-induced activation of Akt, mTOR, and eukaryotic initiation factor 4E binding protein 1. The results showed that Lut prevented Cd-induced cell death partly by blocking the Akt/mTOR signaling pathway. Lut may be a potential agent for preventing Cd-induced nerve cell damage and neurodegenerative diseases.

Defective Mitochondrial Dynamics and Protein Degradation Pathways Underlie Cadmium-Induced Neurotoxicity and Cell Death in Huntington's Disease Striatal Cells.

Exposure to heavy metals, including cadmium (Cd), can induce neurotoxicity and cell death. Cd is abundant in the environment and accumulates in the striatum, the primary brain region selectively affected by Huntington's disease (HD). We have previously reported that mutant huntingtin protein (mHTT) combined with chronic Cd exposure induces oxidative stress and promotes metal dyshomeostasis, resulting in cell death in a striatal cell model of HD. To understand the effect of acute Cd exposure on mitochondrial health and protein degradation pathways, we hypothesized that expression of mHTT coupled with acute Cd exposure would cooperatively alter mitochondrial bioenergetics and protein degradation mechanisms in striatal STHdh cells to reveal novel pathways that augment Cd cytotoxicity and HD pathogenicity. We report that mHTT cells are significantly more susceptible to acute Cd-induced cell death as early as 6 h after 40 µM CdCl2 exposure compared with wild-type (WT). Confocal microscopy, biochemical assays, and immunoblotting analysis revealed that mHTT and acute Cd exposure synergistically impair mitochondrial bioenergetics by reducing mitochondrial potential and cellular ATP levels and down-regulating the essential pro-fusion proteins MFN1 and MFN2. These pathogenic effects triggered cell death. Furthermore, Cd exposure increases the expression of autophagic markers, such as p62, LC3, and ATG5, and reduces the activity of the ubiquitin-proteasome system to promote neurodegeneration in HD striatal cells. Overall, these results reveal a novel mechanism to further establish Cd as a pathogenic neuromodulator in striatal HD cells via Cd-triggered neurotoxicity and cell death mediated by an impairment in mitochondrial bioenergetics and autophagy with subsequent alteration in protein degradation pathways.

Neuroprotection of resveratrol against cadmium-poisoning acts through dual inhibition of mTORC1/2 signaling

Resveratrol is a natural polyphenol with neuroprotective function. The underlying mechanism is not well understood. Our previous studies have identified that resveratrol antagonizes cadmium (Cd) neurotoxicity via targeting PP2A/PP5-mediated Erk1/2 and JNK pathways. Here we show that resveratrol protected against Cd-poisoning also by blocking Cd-induced activation of mTORC1 and mTORC2 pathways in PC12 cells and murine primary neurons. Co-treatment with inhibitors of mTORC1 (rapamycin), mTORC1/2 (PP242), Erk1/2 (U0126) and/or JNK (SP600125), knockdown of mTOR, or disruption of mTORC1 and/or mTORC2 by silencing raptor, rictor or raptor/rictor, respectively, markedly potentiated the inhibitory effects of resveratrol on Cd-induced phosphorylation of S6K1/4E-BP1 (mTORC1 substrates), Akt (mTORC2 substrate), Erk1/2 and/or JNK/c-Jun, cleavage of caspase-3 and cell death in PC12 cells and/or primary neurons. Knockdown of S6K1 or 4E-BP1, or ectopic expression of constitutively hypophosphorylated 4E-BP1 (4E-BP1-5A) reinforced the resveratrol's inhibition on Cd-evoked cell death, whereas ectopic expression of constitutively active S6K1 or knockdown of 4E-BP1 attenuated the resveratrol's inhibition on Cd-induced cell death. Co-treatment with Akt inhibitor or overexpression of dominant negative Akt (dn-Akt) strengthened the resveratrol's suppression on Cd-induced ROS, Erk1/2 activation and apoptosis, whereas overexpression of constitutively active Akt (myr-Akt) conferred high resistance to the resveratrol's inhibitory effects in the neuronal cells. Taken together, the results indicate that resveratrol attenuates Cd-induced neuronal apoptosis partly through inhibition of mTORC1/2 pathways. Our studies highlight that resveratrol can be exploited for the prevention of Cd toxicity related to neurodegenerative diseases.

Synoviocytes from pigmented villonodular synovitis are less sensitive to cadmium-induced cell death than synoviocytes from rheumatoid arthritis

Pigmented villonodular synovitis (PVNS) is a rare inflammatory articular disease sharing common characteristics with rheumatoid arthritis (RA), notably hyperplasia of the synovium due to a hyperproliferation of synoviocytes, and with cancer owing to mutations of the CSF1/M-CCSF gene. Targeting synovium hyperplasia by the local delivery of Cadmium (Cd) has been already tested in vitro and in vivo models of RA and could be applied to PVNS. PVNS and RA synoviocytes were exposed to low doses of Cd. After different culture time points, a qualitative analysis was done by microscopy and quantitative measurements of apoptosis, cell viability and IL-6 production were carried. IL-6 production by PVNS synovial tissue was also quantified after Cd treatment with or without the presence of pro-inflammatory cytokines (IL-17 + TNF). Addition of Cd induced cell death in both PVNS (1 ppm) and RA (0.1 ppm) synoviocytes, which increased with time and Cd concentrations. Cd increased the percentage of apoptotic cells and decreased cell viability and IL-6 production. In all these experiments, PVNS synoviocytes were tenfold less sensitive to Cd than RA synoviocytes. Cd decreased IL-6 production by PVNS synovial tissue and its effect was enhanced with pro-inflammatory cytokines. In summary, PVNS synoviocytes show resistance to Cd-induced cell death and decreased inflammation. Intra-articular use of Cd could represent a potential therapeutic tool in PVNS.

Clerodane diterpene induces apoptosis/anoikis and suppresses migration and invasion of human bladder cancer cells through the histone deacetylases, integrin–focal adhesion kinase, and matrix metalloproteinase 9 signalling pathways

Clerodane diterpene, a class of bicyclic diterpenoids, is found in hundreds of plant species. 16-hydroxycleroda-3,13-dien-15,16-olide (CD) can be isolated from the plant Polyalthia longifolia and has been applied against oral cancer and glioma by xenograft model. In this study, we aim to explore its antitumour action by examining its histone deacetylase (HDAC) activity and integrin-associated intracellular signalling pathway on T24 human bladder cancer (BC) cells. Our results revealed that CD-inhibited colony formation, HDAC activity, HDAC (1, 2 and 3) mRNA and cell spreading on fibronectin-coated surfaces in a concentration-dependent manner. Furthermore, decreased cFLIP and increased caspase-8 cleavage accompanied CD-induced cell death. At non-toxic concentrations, CD blocked the migration and invasion of T24 cells. CD hindered migration and invasion by the downregulation of fibronectin, integrin α5β1, β-catenin, FAK, vinculin and Rho A, as well as by reduction of phosphorylated glycogen synthase kinase 3β (pGSK3β), pSrc, pstat3 and pNFκB. We observed that the MMP9 gene was closely linked with prognosis of patients with bladder cancer. MMP9 protein levels and activity were largely attenuated by CD in a concentration-dependent manner. In conclusion, CD-induced caspase-8-dependent apoptosis and suppressed migration and invasion by blocking several intracellular signalling pathways, including downregulation of HDAC activity and integrin-FAK and MMP9 pathways.

Hydrogen Sulfide Inhibits Cadmium-Induced Cell Death of Cucumber Seedling Root Tips by Protecting Mitochondrial Physiological Function

Hydrogen sulfide (H2S) can alleviate Cd-induced cell death, but the molecular mechanisms are not clear. To shed light on these mechanisms, cell death induced by 200 μM cadmium chloride in cucumber seedlings root tips was used as a model system. Here, we report that the negative effect of Cd stress in mitochondrial physiological functions include changes in cytochrome c/a, mitochondrial membrane permeability transition pores, and adenosine triphosphatase (ATPase). Moreover, Cd stress led to the release of mitochondrial Ca2+ into the cytosol. Exogenous application of sodium hydrosulfide (NaHS, a donor of H2S) inhibited cell death and maintains mitochondrial function by reducing mitochondrial hydrogen peroxide accumulation, increasing ATPase activity and down-regulating CsVDAC and CsANT expression. In summary, H2S suppressed Cd-induced cell death by improving mitochondrial physiological properties.

Endoplasmic reticulum stress-mediated autophagy activation is involved in cadmium-induced ferroptosis of renal tubular epithelial cells

Acute cadmium (Cd) exposure is a significant risk factor for renal injury and lacks effective treatment strategies. Ferroptosis is a recently identified iron-dependent form of nonapoptotic cell death mediated by membrane damage resulting from lipid peroxidation, and it is implicated in many diseases. However, whether ferroptosis is involved in Cd-induced renal injury and, if so, how it operates. Here, we show that Cd can induce ferroptosis in kidney and renal tubular epithelial cells, as demonstrated by elevation of intracellular iron levels and lipid peroxidation, as well as impaired antioxidant production. Treatment with a ferroptosis inhibitor alleviated Cd-induced cell death. Intriguingly, we established that Cd-induced ferroptosis depended on endoplasmic reticulum (ER) stress, by demonstrating that Cd activated the PERK-eIF2α-ATF4-CHOP pathway and that inhibition of ER stress reduced ferroptosis caused by Cd. We further found that autophagy was required for Cd-induced ferroptosis because the inhibition of autophagy by chloroquine mitigated Cd-induced ferroptosis. Furthermore, we showed that iron dysregulation by ferritinophagy contributed to Cd-induced ferroptosis, by showing that the iron chelator desferrioxamine alleviated Cd-induced cell death and lipid peroxidation. In addition, ER stress is likely activated by MitoROS which trigger autophagy and ferroptosis. Collectively, our results indicate that ferroptosis is involved in Cd-induced renal toxicity and regulated by the MitoROS-ER stress-ferritinophagy axis.

Hydrogen sulfide negatively regulates cd-induced cell death in cucumber (Cucumis sativus L) root tip cells

BackgroundHydrogen sulfide (H2S) is a gas signal molecule involved in regulating plants tolerance to heavy metals stress. In this study, we investigated the role of H2S in cadmium-(Cd-) induced cell death of root tips of cucumber seedlings.ResultsThe results showed that the application of 200 μM Cd caused cell death, increased the content of reactive oxygen species (ROS), chromatin condensation, the release of Cytochrome c (Cyt c) from mitochondria and activated caspase-3-like protease. Pretreatment of seedlings with 100 μM sodium hydrogen sulfide (NaHS, a H2S donor) effectively alleviated the growth inhibition and reduced cell death of root tips caused by Cd stress. Additionally, NaHS + Cd treatment could decrease the ROS level and enhanced antioxidant enzyme activity. Pretreatment with NaHS also inhibited the release of Cyt c from the mitochondria, the opening of the mitochondrial permeability transition pore (MPTP), and the activity of caspase-3-like protease in the root tips of cucumber seedling under Cd stress.ConclusionH2S inhibited Cd-induced cell death in cucumber root tips by reducing ROS accumulation, activating the antioxidant system, inhibiting mitochondrial Cyt c release and reducing the opening of the MPTP. The results suggest that H2S is a negative regulator of Cd-induced cell death in the root tips of cucumber seedling.

Indole-3-butyric acid priming reduced cadmium toxicity in barley root tip via NO generation and enhanced glutathione peroxidase activity.

Activation of GPX and enhanced NO level play a key role in IBA-mediated enhanced Cd tolerance in young barley roots. Application of exogenous indole-3-acetic acid (IAA) or an IAA precursor improves the tolerance of plants to heavy metals. However, the physiology of these tolerance mechanisms remains largely unknown. Therefore, we studied the priming effect of indole-3-butyric acid (IBA), an IAA precursor, on mild and severe cadmium (Cd) stress-induced responses in roots of young barley seedlings. IBA, similarly to mild Cd stress, significantly increased the glutathione peroxidase (GPX) activity in the apexes of barley roots, which remained elevated after the IBA pretreatment as well. IBA pretreatment-evoked high nitric oxide generation in roots effectively reduced the high superoxide level under the severe Cd stress, leading to less toxic peroxynitrite accumulation accompanied by markedly reduced Cd-induced cell death. On the other hand, the IBA-evoked changes in IAA homeostasis resulted in root growth reorientation from longitudinal elongation to radial swelling. However, the application of an IAA signaling inhibitor, following the activation of defense responses by IBA, was able to promote root growth even at high concentrations of Cd. Based on the results, it can be concluded that the application of IBA, as an effective activator of Cd tolerance mechanisms in young barley roots, and the subsequent use of an IAA signaling inhibitor for the inhibition of root morphogenic responses induced by altered auxin metabolism, results in a high degree of root Cd tolerance, helping it to withstand even the transient exposure to lethal Cd concentration without the absolute inhibition of root growth.

16-Hydroxycleroda-3,13-Dien-15,16-Olide Induces Apoptosis in Human Bladder Cancer Cells through Cell Cycle Arrest, Mitochondria ROS Overproduction, and Inactivation of EGFR-Related Signalling Pathways

A clerodane diterpene compound 16-hydroxycleroda-3,13-dien-15,16-olide (CD) is considered a therapeutic agent with pharmacological activities. The present study investigated the mechanisms of CD-induced apoptosis in T24 human bladder cancer cells. CD inhibited cell proliferation in a concentration and time-dependent manner. CD-induced overproduction of reactive oxygen species and reduced mitochondrial membrane potential, associated with reduced expression of Bcl-2 and increased levels of cytosolic cytochrome c, cleaved PARP-1 and caspase-3. In addition, CD treatment led to cell cycle arrest at the G0/G1 phase and inhibited expression of cyclin D1 and cyclin-dependent kinases 2 and 4 and led to increased levels of p21, p27Kip1 and p53. All of these events were accompanied with a reduction of pEGFR, pMEK1/2, pERK1/2, pAkt, pmTOR, pP70S6K1, HIF-1α, c-Myc and VEGF. RNAseq-based analysis revealed that CD-induced cell death was characterised by an increased expression of stress and apoptotic-related genes as well as inhibition of the cell cycle-related genes. In summary, CD induces apoptosis in T24 bladder cancer cells through targeting multiple intracellular signaling pathways as a result of oxidative stress and cell cycle arrest.

MhNRAMP1 From Malus hupehensis Exacerbates Cell Death by Accelerating Cd Uptake in Tobacco and Apple Calli.

Excessive cadmium (Cd) damages plants by causing cell death. The present study discusses the function of natural resistance-associated macrophage protein (NRAMP) on cell death caused by Cd in Malus hupehensis. MhNRAMP1 was isolated from M. hupehensis roots, and its protein was located in the cell membrane as a transmembrane protein characterized by hydrophobicity. MhNRAMP1 expression in the roots was induced by Cd stress and calcium (Ca) deficiency. MhNRAMP1 overexpression increased Cd concentration in yeasts and enhanced their sensitivity to Cd. Phenotypic comparisons of plants under Cd stress revealed that the growth of transgenic tobacco and apple calli overexpressing MhNRAMP1 was worse than that of the wild type (WT). The Cd2+ influx of transgenic tobacco roots and apple calli was higher, and the recovery time of the Cd2+ influx to a stable state in transgenic apple calli was longer than that of the WT. Cd accumulation and the percentage of apoptotic cells in transgenic lines were higher. Correspondingly, the caspase-1-like and vacuolar processing enzyme (VPE) activities and MdVPEγ expression were higher in transgenic apple calli, but the expression levels of genes that inhibit cell death were lower than those in the WT under Cd stress. Moreover, the Cd translocation from the roots to leaves was increased after MhNRAMP1 overexpression, but the Cd translocation from the leaves to seeds was not affected. These results suggest that MhNRMAP1 exacerbated Cd-induced cell death, which was accomplished by mediating Cd2+ uptake and accumulation, as well as stimulating VPE.

Cytoplasmic sirtuin 6 translocation mediated by p62 polyubiquitination plays a critical role in cadmium-induced kidney toxicity.

Sirtuin 6 (Sirt6) is important for maintaining kidney homeostasis and function. Cd exposure increases the risk of developing kidney diseases. However, the role of Sirt6 in kidney disease mechanisms is unclear. Here, we evaluated the role of Sirt6 in Cd-induced kidney toxicity. After Cd exposure, p62/sequestosome-1 (SQSTM1), an autophagy substrate, accumulated in mouse kidney mesangial cells in monomeric and polyubiquitinated (polyUb) forms. Sirt6 accumulated in response to Cd treatment at concentrations below the half-maximal inhibitory concentration and decreased after 12h of treatment. Sirt6 and p62 co-localized in the nucleus and redistributed to the cytosol after Cd treatment. Sirt6 was mainly present in nuclei-rich membrane fractions. Sirt6 interacted with p62. Ub, and microtubule-associated protein light chain 3 (LC3). Knockdown of p62 promoted Sirt6 nuclear accumulation and inhibited apoptosis. Sirt6 overexpression altered levels of polyUb-p62 and apoptosis. At earlier times during Cd treatment, polyubiquitination of p62 and apoptosis were reduced. Cytoplasmic translocation of Sirt6 occurred later, with increased polyubiquitination of p62 and apoptosis. Bafilomycin 1 (BaF1) treatment promoted cytosolic Sirt6 accumulation, increasing cell death. Silencing autophagy related 5 (Atg5) increased nuclear Sirt6 levels, reduced polyUb-p62, and inhibited cell death, indicating that autophagy was necessary for Sirt6 redistribution. Cd resistance was associated with reduced polyUb-p62 and persistent Sirt6 expression. Cd treatment in mice for 4weeks promoted p62, Sirt6, and LC3-II accumulation, inducing apoptosis in kidney tissues. Overall, our findings show that polyUb-p62 targeted Sirt6 to autophagosomes, playing a crucial role in Cd-induced cell death and kidney damage.

Metformin attenuates cadmium-induced neuronal apoptosis in vitro via blocking ROS-dependent PP5/AMPK-JNK signaling pathway

Cadmium (Cd), a toxic environment contaminant, induces reactive oxygen species (ROS)-mediated neuronal apoptosis and consequential neurodegenerative disorders. Metformin, an anti-diabetic drug, has recently received a great attention owing to its protection against neurodegenerative diseases. However, little is known regarding the effect of metformin on Cd-induced neurotoxicity. Here we show that metformin effectively prevented Cd-evoked apoptotic cell death in neuronal cells, by suppressing Cd activation of c-Jun N-terminal kinases (JNK), which was attributed to blocking Cd inactivation of protein phosphatase 5 (PP5) and AMP-activated protein kinase (AMPK). Inhibition of JNK with SP600125, knockdown of c-Jun, or overexpression of PP5 potentiated metformin's inhibitory effect on Cd-induced phosphorylation of JNK/c-Jun and apoptosis. Activation of AMPK with AICAR or ectopic expression of constitutively active AMPKα strengthened the inhibitory effects of metformin on Cd-induced phosphorylation of JNK/c-Jun and apoptosis, whereas expression of dominant negative AMPKα weakened these effects of metformin. Metformin repressed Cd-induced ROS, thereby diminishing cell death. N-acetyl-l-cysteine enhanced the inhibitory effects of metformin on Cd-induced ROS and apoptosis. Moreover, using Mito-TEMPO, we further demonstrated that metformin attenuated Cd-induced cell death by suppressing induction of mitochondrial ROS. Taken together, these results indicate that metformin prevents mitochondrial ROS inactivation of PP5 and AMPK, thus attenuating Cd-induced JNK activation and apoptosis in neuronal cells. Our data highlight that metformin may be a promising drug for prevention of Cd-induced oxidative stress and neurodegenerative diseases.

Cadmium induces cytotoxicity in normal mouse renal MM55.K cells

ABSTRACT The toxicity of cadmium (Cd) occurs through accumulation in the environment. The precise mechanism underlying Cd toxicity remains unclear. Therefore, in the present study, we studied the effects of Cd on MM55.K cells and investigated the mechanisms underlying Cd-induced cell death. CdCl2 significantly elevated apoptotic cell death, mitochondrial membrane potential (ΔΨm) loss, and caspase-dependent cell death. Moreover, immunoblotting results revealed that CdCl2 down-regulated the inhibitor of apoptotic protein such as survivin and Bcl-2 which led to the activation of caspase-3 and the cleavage of PARP in MM55.K cells. Besides, CdCl2 caused the up-regulation of ROS-related proteins such as HO-1 and ER stress-related proteins such as GRP78 and CHOP in MM55.K cells. CdCl2 toxicity resulted in the down-regulation of the AKT pathway that leads to the up-regulation of phosphorylated JNK and p38 in MM55.K cells. Thus, CdCl2 induce toxicity by AKT/MAPK regulation and causing ROS production, ER stress, ΔΨm loss, and apoptotic cell death in normal mouse renal cells.

Effects of combined stressors to cadmium and high temperature on antioxidant defense, apoptotic cell death, and DNA methylation in zebrafish (Danio rerio) embryos.

Fish are frequently affected by environmental stressors, such as temperature changes and heavy metal exposure, in aquatic ecosystems. In this study, we evaluated the combined effects of cadmium (Cd) toxicity and temperature (rearing temperature of 26°C and heat stress at 34°C) on zebrafish (Danio rerio) embryos. The survival and heart rates of zebrafish embryos decreased at relatively high Cd concentrations of 0.07 and 0.1mgL-1. Abnormal morphology was induced by exposure to a combination of Cd toxicity and heat stress. The yolk sac edema size was not significantly different between the control- and Cd-treated groups. Cd exposure induced reactive oxygen species (ROS) production and cell death in the live zebrafish. High temperature (34°C) triggered Cd-induced cell death and intracellular ROS production to a greater extent than the rearing temperature of 26°C. Transcriptional levels of six genes-CAT, SOD, p53, BAX, Dnmt1, and Dnmt3b-were investigated. The mRNA expression of CAT and SOD, molecular indicators of oxidative stress, was increased significantly at 34°C after Cd exposure. The mRNA expression of CAT was more sensitive to temperature than that of SOD in Cd-treated zebrafish. p53 and BAX, apoptosis-related genes, were upregulated upon combined exposure to high temperature and Cd. In addition, at 34°C, the expression of Dnmt1 and Dnmt3b transcripts, markers of DNA methylation, was increased upon exposure of zebrafish to all concentrations of Cd. Overall, these results suggest that high temperature facilitates the potential role of Cd toxicity in the transcriptional regulation of genes involved in the antioxidant system, apoptosis, and DNA methylation.

Selenite antagonizes the phytotoxicity of Cd in the cattail Typha angustifolia.

The Phytotoxicity of and mechanism underlying selenite-mediated tolerance to Cd stress in Typha angustifolia were studied hydroponically with respect to metal uptake and translocation, photosynthesis-related parameters, contents of proline and O2•-, products of lipid peroxidation, cell viability, enzymatic and non-enzymatic antioxidants, glyoxalases and phytochelatins. T. angustifolia were exposed to 25, 50 and 100μM of Cd alone and in conjunction with 5mgL-1 of selenite in full-strength Hoagland's nutrient solution for 30 days. Results showed that Cd contents in T. angustifolia leaves and roots increased in a dose-dependent manner and were higher in roots, but those of BAC, BCF and TF changed in a contrary pattern. Addition of selenite to Cd-containing treatments further reduced Cd levels in T. angustifolia leaves and roots, as well as BAC, BCF and TF. A diphasic effect was found in T. angustifolia for the contents of total chlorophyll, GSH, PC and GSSG, as well as activities of CAT, POD, SOD and GR, in response to Cd stress alone and in conjunction with selenite supplementation, but the same effect was not observed for Pn, Cond, Tr, Ci, Fv/Fm and ϕPSII. In contrast, exogenous selenite supplementation enhanced the contents of total chlorophyll and the non-enzymatic antioxidants, as well as activities of enzymatic antioxidants, while the values of photosynthetic fluorescence parameters were rescued. Selenite addition decreased Cd-induced cell death. Proline contents and Gly I activities in T. angustifolia leaves kept increasing in a dose-dependent manner of Cd concentrations in the growth media and selenite addition further enhanced both parameters. Addition of selenite could quench Cd-mediated generation of MDA, O2•- and MG in T. angustifolia leaves and reduce Cd-induced Gly II activity. A U-shaped GSH/GSSG ratio in T. angustifolia leaves suggests a possible trade-off between PC synthesis and GR activity since both share the same substrate GSH. Therefore, confined BAC, BCF and TF were a mechanism that confers T. angustifolia tolerance to Cd stress, and that exogenous selenite supplementation could depress Cd-induced stress in T. angustifolia by rescuing the photosynthetic fluorescence, enhancing non-enzymatic and enzymatic antioxidants that scavenge O2•- and MG, and potentiating PC synthesis that chelates Cd.

Cd2+ uptake inhibited by MhNCED3 from Malus hupehensis alleviates Cd-induced cell death

Abscisic acid (ABA) is a plant hormone induced by cadmium (Cd) stress. This study discusses the role of ABA biosynthesis mediated by 9-cis-epoxycarotenoid dioxygenase (MhNCED3) in cell death caused by Cd. Results show that ABA application to Cd-stressed Malus hupehensis reduced the Cd2+ influx to roots and Cd accumulation. The apoptosis rate of root cells was also decreased after ABA application, and the expression of vacuolar processing enzyme (MhγVPE) and VPE activity was down-regulated. Transgenic Arabidopsis and apple calli with ectopic-expressed MhNCED3 had lower Cd2+ influx, lower Cd contents, as well as lower expression of genes (NRAMP and IRT) that control Cd2+ uptake under Cd stress. The cell death in overexpressed MhNCED3 apple calli was lower than that in the wild type. The expression of γVPE and VPE activity also showed a lower level in transgenic plants. The growth of transgenic apple calli that had a higher endogenous ABA level was expectedly better than that of the wild type under Cd stress. Additionally, ABA restricted the upward-translocation of Cd, with evidence of a lower translocation factor and expression of the gene involved in Cd delivery (AtHMA2). Our results suggested that MhNCED3 regulated Cd-induced cell death via VPE, which was probably accomplished through the Cd uptake inhibited by ABA.

Enhanced nitric oxide generation mitigates cadmium toxicity via superoxide scavenging leading to the formation of peroxynitrite in barley root tip

The aim of this study was to observe the possible function of increased superoxide and NO production in the response of barley root tip to the harmful level of Cd. While superoxide generation was detected only in the transition zone, the formation of NO was observed in the apical elongation zones of the control root tips. However, the root region with the superoxide generation was also associated with peroxynitrite specific fluorescence signal. Superoxide, H2O2 and peroxynitrite generation increased with Cd treatment in a dose-dependent manner. In turn, NO level increased at low 10–20 μM but decreased at high 50–60 μM Cd concentrations in comparison with the control. While co-treatment of roots with rotenone markedly attenuated the Cd-induced superoxide generation and lipid peroxidation, it increased the level of NO in the root tips. Although rotenone did not influence the Cd-induced increase of GPX activity at 10–30 μM Cd concentrations, it markedly reversed the high 40–60 μM Cd concentrations-induced decline of GPX activity. Cd-induced cell death was associated with robust superoxide generation, but not with a high level of peroxynitrite. The Cd-evoked inhibition of root growth was significantly reversed by a strong antioxidant N-acetyl cysteine but not by a peroxynitrite scavenger uric acid, suggesting that similarly to Cd-induced cell death, an imbalance in the ROS homeostasis and not an enhanced level of peroxynitrite is responsible for the Cd-induced root growth inhibition. Based on these findings, it can be assumed that NO acts mainly in the regulation of superoxide level in the tips of root. Under Cd stress, the enhanced NO level is involved in the scavenging of highly toxic superoxide through the formation of peroxynitrite, thus reducing the superoxide-mediated cell death in barley root.

Cytoprotective effects of taxifolin against cadmium-induced apoptosis in human keratinocytes.

Cadmium (Cd) is a heavy metal widely used in industry, and the skin is an important target of this metal. Taxifolin (Tax), a natural source of bioflavonoids found in various conifers, exerts multiple biologic effects on skin cells. However, the mechanisms by which Tax protects keratinocytes against Cd are currently unclear. We investigated the cytoprotective effects of Tax against Cd-induced apoptosis in the human HaCaT keratinocyte. The water-soluble tetrazolium salt (WST-1) assay and Annexin V/propidium iodide double-staining assay results showed that Cd-induced cell death was lower in cells treated with Tax (0-100 μM) than in cells treated with Cd alone. Additionally, a reduction of Cd-induced DNA fragmentation by Tax was shown by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay. The levels of reactive oxygen species were also lower in Cd/Tax-treated cells than in Cd-treated cells. We employed a two-dimensional electrophoresis-based proteomic analysis to identify treatment-related alterations in protein expression. Tax downregulated cathepsin B and D and upregulated hsp27, cyclophilin A, and peroxiredowin-1. Western blotting confirmed the downregulation of cathepsin B and D and the upregulation of hsp27. The cytoprotective effects of Tax against Cd-induced apoptosis were also characterized by the changes in the activity of caspase 3, -7, poly ADP-ribose polymerase, the cellular proliferation-related ERK1/2, and AKT. Furthermore, the levels of cell cycle-related proteins, such as SP1 and p21, decreased, whereas p53 level increased. We concluded that Tax reduced Cd cytotoxicity and Cd-induced apoptosis by inhibiting the apoptotic pathway.

GSK-3\u03b2-mediated regulation of cadmium-induced cell death and survival

BackgroundPrevious studies indicated that cadmium (Cd) increases PI3-kinase/Akt phosphorylation, resulting in an alteration in GSK-3β activity. However, the mechanism of Cd-induced endoplasmic reticulum (ER) stress in neuronal cells has yet to be studied in needs further elucidation. We examined the role of GSK-3β in Cd-induced neuronal cell death and the related downstream signaling pathways.MethodsSH-SY5Y human neuroblastoma cells were treated with 10 or 20 μM BAPTA-AM and 1 μM wortmannin for 30 min and then incubated with 25 μM Cd for 12 h. Apoptotic cells were visualized via DAPI and PI staining. Data were evaluated with one-way analysis of variance (ANOVA) followed by Student’s t-test. Data are expressed as the means ± SD of experiments performed at least three times.ResultsTreatment of human neuronal SH-SY5Y cells with Cd induced ER, stress as evidenced by the increased expression of GRP78, which is a marker of ER stress. Cd exposure significantly increased the phosphorylation of Akt at thr308 and ser473 and that of GSK-3β at ser9 in a time-dependent manner, while the total protein levels of GSK-3β and Akt did not change. Cd-induced apoptosis was higher in GSK-3β-knockdown cells than in normal cells.ConclusionsOur data suggest that Akt/GSK-3β signaling activated by Cd is involved in neuronal cell survival.