Membraneous localization of Jpk is not essential to exert cytotoxicity in F9 teratocarcinoma cells

A novel gene, Jopock (Jpk), which was isolated as a trans-acting factor associating with the PSRE of murine Hoxa-7, has been shown to be toxic to both prokaryotic and eukaryotic cells when overexpressed. Here we demonstrate that the overexpression of enhanced green fluorescent protein (EGFP)-tagged Jpk in F9 cells results in the induction of apoptosis, as indicated by phosphatidylserine exposure, DNA fragmentation, and the alteration of mitochondria transmembrane potential. Fluorescence microscopy showed that EGFP-fused Jpk was mainly localized in the endoplasmic reticulum (ER) and a small amount was found in the mitochondria. Deletion mutants with a transmembrane (TM) domain showed a distribution similar to that of EGFP-Jpk, whereas constructs with a deletion of the TM domain localized in the whole cells. Deletion mapping experiments showed that Jpk with an N-terminal part deleted stimulated apoptosis to almost the same extent as that of the wild-type Jpk, indicating that the localization of Jpk in the ER and the TM domain does not appear to be essential for inducing cytotoxicity. Overall, these results suggest that Jpk, particularly the C-terminal part of Jpk and/or 3'UTR, triggers apoptosis through a perturbation of mitochondrial membrane permeabilization.     

Effects of Boron Supplementation on Peripartum Dairy Cows’ Health

In the present study, toxicity of commercial zinc oxide nanoparticles (ZnO NPs) was studied on the bacterium Pseudomonas sp., human promyelocytic leukemia (HL-60) cells, and peripheral blood mononuclear cells (PBMC). The toxicity was assessed by measuring growth, cell viability, and protein expression in bacterial cell. The bacterial growth and viability decreased with increasing concentrations of ZnO NP. Three major proteins, ribosomal protein L1 and L9 along with alkyl hydroperoxides reductase, were upregulated by 1.5-, 1.7-, and 2.0-fold, respectively, after ZnO NP exposure. The results indicated oxidative stress as the leading cause of toxic effect in bacteria. In HL-60 cells, cytotoxic and genotoxic effects along with antioxidant enzyme activity and reactive oxygen species (ROS) generation were studied upon ZnO NP treatment. ZnO NP exhibited dose-dependent increase in cell death after 24-h exposure. The DNA-damaging potential of ZnO NP in HL-60 cells was maximum at 0.05 mg/L concentration. Comet assay showed 70–80% increase in tail DNA at 0.025 to 0.05 mg/L ZnO NP concentration. A significant increase of 1.6-, 1.4-, and 2.0-fold in ROS level was observed after 12 h. Genotoxic potential of ZnO NPs was also demonstrated in PBMC through DNA fragmentation. Thus, ZnO NP, besides being an essential element having antibacterial activity, also showed toxicity towards human cells (HL-60 and PBMC).     

Plumbagin and juglone induce caspase-3-dependent apoptosis involving the mitochondria through ROS generation in human peripheral blood lymphocytes

The phytochemicals plumbagin and juglone have recently been gaining importance because of their various pharmacological activities. In this study, these compounds are shown to induce concentration- and time-dependent toxicity in human peripheral blood lymphocytes via the apoptotic pathway. Flow cytometry data revealed the occurrence of about 28% early apoptotic cells after 6 h exposure to 10 μM plumbagin and 35% late apoptotic cells and about 43% sub-G1 population after 24 h. The cytotoxic effect of plumbagin was at least twofold higher than that of juglone as evidenced by the IC₅₀ value for cytotoxicity. Characteristic apoptotic features such as chromatin condensation and apoptotic body formation were observed through TEM, and membrane blebbing and cell surface smoothening were seen in SEM studies. Generation of ROS was evidenced through the HPLC analysis of superoxide-specific 2-OH-E+ formation. In addition, a decrease in GSH levels parallel to ROS production was observed. Reversal of apoptosis in both NAC- and Tempol-pretreated cells indicates the involvement of both ROS generation and GSH depletion in plumbagin- and juglone-induced apoptosis. The mechanistic pathway involves a decrease in MMP; alterations in the levels of Bcl-2, Bax, and cytosolic cytochrome c; and PARP-1 cleavage subsequent to caspase-3 activation.     

The influence of reactive oxygen species on cell cycle progression in mammalian cells

Cell cycle regulation is performed by cyclins and cyclin dependent kinases (CDKs). Recently, it has become clear that reactive oxygen species (ROS) influence the presence and activity of these enzymes and thereby control cell cycle progression. In this review, we first describe the discovery of enzymes specialized in ROS production: the NADPH oxidase (NOX) complexes. This discovery led to the recognition of ROS as essential players in many cellular processes, including cell cycle progression. ROS influence cell cycle progression in a context-dependent manner via phosphorylation and ubiquitination of CDKs and cell cycle regulatory molecules. We show that ROS often regulate ubiquitination via intermediate phosphorylation and that phosphorylation is thus the major regulatory mechanism influenced by ROS. In addition, ROS have recently been shown to be able to activate growth factor receptors. We will illustrate the diverse roles of ROS as mediators in cell cycle regulation by incorporating phosphorylation, ubiquitination and receptor activation in a model of cell cycle regulation involving EGF-receptor activation. We conclude that ROS can no longer be ignored when studying cell cycle progression.     

Isoalantolactone induces reactive oxygen species mediated apoptosis in pancreatic carcinoma PANC-1 cells

Isoalantolactone, a sesquiterpene lactone compound possesses antifungal, antibacteria, antihelminthic and antiproliferative activities. In the present study, we found that isoalantolactone inhibits growth and induces apoptosis in pancreatic cancer cells. Further mechanistic studies revealed that induction of apoptosis is associated with increased generation of reactive oxygen species, cardiolipin oxidation, reduced mitochondrial membrane potential, release of cytochrome c and cell cycle arrest at S phase. N-Acetyl Cysteine (NAC), a specific ROS inhibitor restored cell viability and completely blocked isoalantolactone-mediated apoptosis in PANC-1 cells indicating that ROS are involved in isoalantolactone-mediated apoptosis. Western blot study showed that isoalantolactone increased the expression of phosphorylated p38 MAPK, Bax, and cleaved caspase-3 and decreased the expression of Bcl-2 in a dose-dependent manner. No change in expression of phosphorylated p38 MAPK and Bax was found when cells were treated with isoalantolactone in the presence of NAC, indicating that activation of these proteins is directly dependent on ROS generation. The present study provides evidence for the first time that isoalantolactone induces ROS-dependent apoptosis through intrinsic pathway. Furthermore, our in vivo toxicity study demonstrated that isoalantolactone did not induce any acute or chronic toxicity in liver and kidneys of CD1 mice at dose of 100 mg/kg body weight. Therefore, isoalantolactone may be a safe chemotherapeutic candidate for the treatment of human pancreatic carcinoma.     

硫氧还蛋白-过氧化物氧还蛋白与过氧化氢构成环路参与肿瘤的发生与发展

组织细胞可经过多种途径产生氧自由基（ROS）,而肿瘤组织由于多种应激因素会产生大量ROS,其中最重要的是过氧化氢（H2O2).H2O2对细胞发挥着致损伤及亚毒性信使的双重作用,作为信使其不仅参与调节正常细胞信号通路,重要的是促进肿瘤的发生及进展. ROS作为一种应激刺激信号激活细胞内的AP-1（activator protein 1）、Nrf-2（NF-E2-related factor 2）等核转录因子,活化后的AP-1、Nrf-2会结合到硫氧还蛋白（sulfiredoxin, SRX）基因启动子上游的调控序列,促进SRX基因的表达.SRX的表达上调则影响其下游的抗氧化蛋白,即特定亚型的过氧化物氧还蛋白（peroxiredoxin, PRX）的活性状态,最终使细胞内H2O2浓度受到调节. 由SRX-PRX轴与H2O2形成1个环路,通过调节H2O2含量来参与细胞众多信号通路.本文对H2O2、SRX及PRX各自的功能进行综述,还进一步探讨三者构成的信号环路对肿瘤的调控机制,从而了解该环路在肿瘤发生发展中所发挥的作用.     

Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including beta-catenin and the NF-kappaB inhibitor IkappaB-alpha. Ubiquitination of these proteins as well as others is catalyzed by the SCF(betaTrCP) ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-kappaB and beta-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin-proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.     

Complexation of the fungal metabolite tenuazonic acid with copper (II), iron (III), nickel (II), and magnesium (II) ions

Tenuazonic acid (TA) is a phytotoxin produced by a fungal pathogen of rice, Pyricularia oryzae. We have synthesized and characterized the metal complexes of TA with copper (II), iron (III), nickel (II), and magnesium (II). The stoichiometry of the complexes determined by microanalysis and mass spectroscopy (D/CI) are Cu(II)TA2, Fe(III)TA3, Ni(II)TA2, and Mg(TA)2. Voltammograms of Fe(III)TA3, and Cu(II)TA2 in methanolic solutions confirmed this stoichiometry. Ni(II)TA2 paramagnetism and visible absorption data suggest an octahedral geometry. Fe(III)TA3 showed a characteristic visible absorption at 450 nm. Addition of Fe(III)Cl3 and Mg(II)Cl2 did not reverse the toxicity of NaTA to rice and bacterial cells, showing that this toxicity is not due to the privation of the cells of these metals essential for cell growth.     

Organometallic iron(III)-salophene exerts cytotoxic properties in neuroblastoma cells via MAPK activation and ROS generation

The objective of the present study was to investigate the specific effects of Iron(III)-salophene (Fe-SP) on viability, morphology, proliferation, cell cycle progression, ROS generation and pro-apoptotic MAPK activation in neuroblastoma (NB) cells. A NCI-DTP cancer screen revealed that Fe-SP displayed high toxicity against cell lines of different tumor origin but not tumor type-specificity. In a viability screen Fe-SP exhibited high cytotoxicity against all three NB cell lines tested. The compound caused cell cycle arrest in G1 phase, suppression of cells progressing through S phase, morphological changes, disruption of the mitochondrial membrane depolarization potential, induction of apoptotic markers as well as p38 and JNK MAPK activation, DNA degradation, and elevated generation of reactive oxygen species (ROS) in SMS-KCNR NB cells. In contrast to Fe-SP, non-complexed salophene or Cu(II)-SP did not raise ROS levels in NB or SKOV-3 ovarian cancer control cells. Cytotoxicity of Fe-SP and activation of caspase-3, -7, PARP, pro-apoptotic p38 and JNK MAPK could be prevented by co-treatment with antioxidants suggesting ROS generation is the primary mechanism of cytotoxic action. We report here that Fe-SP is a potent growth-suppressing and cytotoxic agent for in vitro NB cell lines and, due to its high tolerance in previous animal toxicity studies, a potential therapeutic drug to treat NB tumors in vivo.     

Diversity of amino acid signaling pathways on autophagy regulation: A novel pathway for arginine

Autophagy is the intracellular bulk degradation process to eliminate damaged cellular machinery and to recycle building blocks, and is crucial for cell survival and cell death. Amino acids modulate autophagy in response to nutrient starvation and oxidative stress. We investigated the relevance of reactive oxygen species (ROS) production on the regulation of autophagy using amino acids, both as a mixture and individually, in rat hepatoma H4-II-E cells. Nutrient starvation elevated ROS production and stimulated autophagy. Treatment with complete (CAA), regulatory (RegAA) and non-regulatory (NonRegAA) amino acid mixtures showed significant suppression of ROS production, whereas only CAA and RegAA exhibited significant suppression of autophagy, suggesting a dissociation of the two responses. The effects of individual amino acids were examined. Leucine from RegAA decreased ROS production and suppressed autophagy. However, methionine and proline from RegAA and arginine, cystine and glutamic acid from NonRegAA suppressed autophagy with an opposite increase in ROS production. Other amino acids from the NonRegAA group showed stimulating effects on ROS production without an autophagic response. Arginine’s effect on autophagy suppression was not blocked by rapamycin, indicating an mTOR-independent pathway. Inhibitor studies on arginine-regulated autophagy may indicate the involvement of NO pathway, which is independent from ROS and mTOR pathways.     

Entamoeba histolytica Induces Cell Death of HT29 Colonic Epithelial Cells via NOX1-Derived ROS

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.     

ROS mediated cytotoxicity of porcine adrenocortical cells induced by QdNOs derivatives in vitro

Quinoxaline 1,4-dioxides (QdNOs) derivatives, the potent synthetic antibacterial group used in food-producing animals, are assumed to have pro-oxidant properties. However, how oxidative stress mediated their adrenal toxicity is far from clear. The aim of this study was to assess the ability of three QdNOs, i.e. olaquindox (OLA), mequindox (MEQ), and cyadox (CYA), to produce reactive oxygen species (ROS) and oxidative cell damage in porcine adrenocortical cells. Multiple approaches such as cell activity assay, biochemical detectation, flow cytometry and fluorescent were used to study the integrated role of ROS homeostasis, mitochondrial redox metabolism and cell apoptosis as well as chemical stability of these drugs. The results showed that OLA and MEQ treatment evoked a significant dose and time-dependent cell damage in adrenocortical cells, well CYA displayed much less toxicity. As for the intracellular ROS production, OLA irritated a persistent and utmost release of ROS while MEQ made a similar but weaker reaction. CYA, however, had a short and unstable release of intracellular ROS. On the other hand, quinoxalinine-2-carboxylie acid (QCA), one of the metabolites of OLA and MEQ, did not cause any significant production of ROS and showed relatively lower toxicity than its parents. Moreover, an imbalance in the redox metabolism and mitochondrial membrane damage has been implicated in adrenal toxicity of QdNOs. ROS scavengers partially reversed QdNOs-induced mitochondrial damage, indicating that mitochondria may be a major target and critical for ROS-mediated cell death. In a word, these results suggested that ROS is a key mediator of QdNOs-induced cell death via mitochondria-dependent pathway in adrenocortical cells. The results provide a mechanism approach in understanding the characterize of adrenal damage caused by QdNOs in vitro, which would in turn, help in designing the appropriate therapeutic strategies of these kind of feed additives.     

Death effector domain of a mammalian apoptosis mediator, FADD, induces bacterial cell death

FADD is a mammalian pro-apoptotic mediator consisting of the N-terminal death effector domain (DED) and the C-terminal death domain (DD). The N-terminal 88-residue fragment of murine FADD was defined as the stable structural unit of DED, as determined by proteolytic digestion and conformational analysis. This domain induced bacterial as well as mammalian cell death, whereas the full-length or DD of FADD did not. The Escherichia coli cells expressing FADD-DED showed elongated cell morphology and an increased level of nicked chromosomal DNA and mutation. The lethality of FADD-DED was abolished by co-expression of thioredoxin and superoxide dismutase or relieved by the addition of vitamin E as a reducing agent and under anaerobic growth conditions. The toxicity of FADD-DED was genetically suppressed by various oxidoreductases of E. coli. All these results suggest that the death effector domain of mammalian FADD induced bacterial cell death by enhancing cellular levels of reactive oxygen species (ROS).     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

Glucose-6-Phosphate Dehydrogenase (G6PD)-Deficient Epithelial Cells Are Less Tolerant to Infection by Staphylococcus aureus

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway and provides reducing energy to all cells by maintaining redox balance. The most common clinical manifestations in patients with G6PD deficiency are neonatal jaundice and acute hemolytic anemia. The effects of microbial infection in patients with G6PD deficiency primarily relate to the hemolytic anemia caused by Plasmodium or viral infections and the subsequent medication that is required. We are interested in studying the impact of bacterial infection in G6PD-deficient cells. G6PD knock down A549 lung carcinoma cells, together with the common pathogen Staphylococcus aureus, were employed in our cell infection model. Here, we demonstrate that a lower cell viability was observed among G6PD-deficient cells when compared to scramble controls upon bacterial infection using the MTT assay. A significant increase in the intracellular ROS was detected among S. aureus-infected G6PD-deficient cells by observing dichlorofluorescein (DCF) intensity within cells under a fluorescence microscope and quantifying this signal using flow cytometry. The impairment of ROS removal is predicted to enhance apoptotic activity in G6PD-deficient cells, and this enhanced apoptosis was observed by annexin V/PI staining under a confocal fluorescence microscope and quantified by flow cytometry. A higher expression level of the intrinsic apoptotic initiator caspase-9, as well as the downstream effector caspase-3, was detected by Western blotting analysis of G6PD-deficient cells following bacterial infection. In conclusion, we propose that bacterial infection, perhaps the secreted S. aureus α-hemolysin in this case, promotes the accumulation of intracellular ROS in G6PD-deficient cells. This would trigger a stronger apoptotic activity through the intrinsic pathway thereby reducing cell viability when compared to wild type cells.     

Distinct mechanisms account for β-amyloid toxicity in PC12 and differentiated PC12 neuronal cells

Whether reactive oxygen species (ROS) mediate beta-amyloid (A beta) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by A beta(25-35). The viability of PC12 and dPC12 cells decreased by 30-40% after a 48-hour exposure to 20 microM A beta(25-35). Microscopic examination showed that A beta(25-35) induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 microM) and other antioxidants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of A beta(25-35). Since H(2)O(2) has been proposed to be involved in A beta toxicity, the effects of H(2)O(2) on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be significantly more resistant to H(2)O(2) than naive PC12 cells. These data suggest that ROS may mediate A beta(25-35) toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were elevated during the differentiation of PC12 cells, the baseline levels of ROS in these two model cell types may determine the intracellular mediators for A beta(25-35) toxicity. Therefore, the protective effects of antioxidants against A beta may depend upon the redox state of the cells.     

Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism

Induction of massive apoptosis of hair follicle cells by chemotherapy has been implicated in the pathogenesis of chemotherapy-induced alopecia (CIA), but the underlying mechanisms of regulation are not well understood. The present study investigated the apoptotic effect of cisplatin in human hair follicle dermal papilla cells and HaCaT keratinocytes, and determined the identity and role of specific reactive oxygen species (ROS) involved in the process. Treatment of the cells with cisplatin induced ROS generation and a parallel increase in caspase activation and apoptotic cell death. Inhibition of ROS generation by antioxidants inhibited the apoptotic effect of cisplatin, indicating the role of ROS in the process. Studies using specific ROS scavengers further showed that hydroxyl radical, but not hydrogen peroxide or superoxide anion, is the primary oxidative species responsible for the apoptotic effect of cisplatin. Electron spin resonance studies confirmed the formation of hydroxyl radicals induced by cisplatin. The mechanism by which hydroxyl radical mediates the apoptotic effect of cisplatin was shown to involve down-regulation of the anti-apoptotic protein Bcl-2 through ubiquitin-proteasomal degradation. Bcl-2 was also shown to have a negative regulatory role on hydroxyl radical. Together, our results indicate an essential role of hydroxyl radical in cisplatin-induced cell death of hair follicle cells through Bcl-2 regulation. Since CIA is a major side effect of cisplatin and many other chemotherapeutic agents with no known effective treatments, the knowledge gained from this study could be useful in the design of preventive treatment strategies for CIA through localized therapy without compromising the chemotherapy efficacy.