Kava extract, an herbal alternative for anxiety relief, potentiates acetaminophen-induced cytotoxicity in rat hepatic cells

The widely used over-the-counter analgesic acetaminophen (APAP) is the leading cause of acute liver failure in the United States and due to this high incidence, a recent FDA Advisory Board recommended lowering the maximum dose of APAP. Kava herbal dietary supplements have been implicated in several human liver failure cases leading to the ban of kava-containing products in several Western countries. In the US, the FDA has issued warnings about the potential adverse effects of kava, but kava dietary supplements are still available to consumers. In this study, we tested the potential of kava extract to potentiate APAP-induced hepatocyte cytotoxicity. In rat primary hepatocytes, co-treatment with kava and APAP caused 100% loss of cell viability, while the treatment of kava or APAP alone caused ∼50% and ∼30% loss of cell viability, respectively. APAP-induced glutathione (GSH) depletion was also potentiated by kava. Co-exposure to kava decreased cellular ATP concentrations, increased the formation of reactive oxygen species, and caused mitochondrial damage as indicated by a decrease in mitochondrial membrane potential. In addition, similar findings were obtained from a cultured rat liver cell line, clone-9. These observations indicate that kava potentiates APAP-induced cytotoxicity by increasing the magnitude of GSH depletion, resulting in oxidative stress and mitochondrial dysfunction, ultimately leading to cell death. These results highlight the potential for drug-dietary supplement interactions even with widely used over-the-counter drugs.     

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt,p38, and JNK pathways in rabbit articular chondrocytes

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined.     

7,8-Dihydroxy-4-methylcoumarin induces apoptosis of human lung adenocarcinoma cells by ROS-independent mitochondrial pathway through partial inhibition of ERK/MAPK signaling

Coumarins have attracted intense interest in recent years because they have been identified from natural sources, especially green plants and have diverse pharmacological properties. In this study, we investigated whether 7,8-dihydroxy-4-methylcoumarin (DHMC) caused apoptosis in A549 human non-small cell lung carcinoma cells (NSCLC) and, if so, by what mechanisms. Here, we show that, in A549 human NSCLC cells, DHMC induces apoptosis through mitochondria-mediated caspase-dependent pathway. Although an increase in the levels of reactive oxygen species (ROS) was observed, pre-treatment with antioxidant showed no protective effect against DHMC-induced apoptosis. In addition, our immunoblot data revealed that DHMC treatment led to down-regulation of Bcl-xl, Bax, p21, Cox-2, p53 and upregulation of c-Myc. Results in the present study for the first time suggest that DHMC induces apoptosis in human lung A549 cells through partial inhibition of ERK/MAPK signaling.     

Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803

Roles of oxidative stress and photoinhibition in high light acclimation were studied using a regulatory mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant strain ΔsigCDE contains the stress responsive SigB as the only functional group 2 σ factor. The ?sigCDE strain grew more slowly than the control strain in methyl-viologen-induced oxidative stress. Furthermore, a fluorescence dye detecting H₂O₂, hydroxyl and peroxyl radicals and peroxynitrite, produced a stronger signal in ?sigCDE than in the control strain, and immunological detection of carbonylated residues showed more protein oxidation in ?sigCDE than in the control strain. These results indicate that ?sigCDE suffers from oxidative stress in standard conditions. The oxidative stress may be explained by the findings that ?sigCDE had a low content of glutathione and low amount of Flv3 protein functioning in the Mehler-like reaction. Although ?sigCDE suffers from oxidative stress, up-regulation of photoprotective carotenoids and Flv4, Sll2018, Flv2 proteins protected PSII against light induced damage by quenching singlet oxygen more efficiently in ?sigCDE than in the control strain in visible and in UV-A/B light. However, in UV-C light singlet oxygen is not produced and PSII damage occurred similarly in the ?sigCDE and control strains. According to our results, resistance against the light-induced damage of PSII alone does not lead to high light tolerance of the cells, but in addition efficient protection against oxidative stress would be required.     

Functional expression of plant alternative oxidase decreases antimycin A-induced reactive oxygen species production in human cells

Alternative oxidase (AOX) plays a pivotal role in cyanide-resistance respiration in the mitochondria of plants, fungi and some protists. Here we show that AOX from thermogenic skunk cabbage successfully conferred cyanide resistance to human cells. In galactose medium, HeLa cells with mitochondria-targeted AOX proteins were found to have significantly less reactive oxygen species production in response to antimycin-A exposure, a specific inhibitor of respiratory complex III. These results suggest that skunk cabbage AOX can be used to create an alternative respiration pathway, which might be important for therapy against various mitochondrial diseases.     

Hyperoxia-mediated oxidative stress increases expression of UCP3 mRNA and protein in skeletal muscle

The uncoupling protein-3 (UCP3) is a mitochondrial protein expressed mainly in skeletal muscle. Among several hypotheses for its physiological function, UCP3 has been proposed to prevent excessive production of reactive oxygen species. In the present study, we evaluated the effect of an oxidative stress induced by hyperoxia on UCP3 expression in mouse skeletal muscle and C2C12 myotubes. We found that the hyperoxia-mediated oxidative stress was associated with a 5-fold and 3-fold increase of UCP3 mRNA and protein levels, respectively, in mouse muscle. Hyperoxia also enhanced reactive oxygen species production and UCP3 mRNA expression in C2C12 myotubes. Our findings support the view that both in vivo and in vitro UCP3 may modulate reactive oxygen species production in response to an oxidative stress.     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

Cu,Zn-superoxide dismutase is required for cell wall structure and for tolerance to cell wall-perturbing agents in Saccharomyces cerevisiae

Here we report that deletion of SOD1, the Cu,Zn-superoxide dismutase in Saccharomyces cerevisiae is sensitive to cell wall-perturbing agents, such as Calcofluor white and Congo red. The sensitivity was restored by retransformation with wild type SOD1 or the addition of N-acetylcysteine or reduced glutathione to the medium. Additionally, the accumulation of reactive oxygen species was observed in sod1Δ mutant in the presence of Calcofluor white or Congo red. Cell wall analysis indicated an increase of cell wall chitin and cell wall thickness in sod1Δ mutant compared to wild type. These results indicate a novel direct connection between antioxidative functions and cell wall homeostasis.     

Vitamin K3-2,3-epoxide induction of apoptosis with activation of ROS-dependent ERK and JNK protein phosphorylation in human glioma cells

2-Methyl-1,4-naphthoquinone (menadione or vitamin K3; EPO) and K3-2,3-epoxide (EPO1), but not vitamin K3-3-OH (EPO2), exhibited cytotoxicity that caused DNA fragmentation and chromatin condensation in U87 and C6 cells. EPO1 showed more-potent cytotoxicity than EPO, and the IC₅₀ values of EPO and EPO1 in U87 cells were 37.5 and 15.7 μM, respectively. Activation of caspase 3 enzyme activity with cleavage of caspase 3 protein was detected in EPO1-treated U87 and C6 cells, and the addition of the caspase 3 peptidyl inhibitor, DEVD-FMK, reduced the cytotoxic effect of EPO1. An increase in the intracellular ROS level by EPO1 was observed in the DCHF-DA analysis, and EPO1-induced apoptosis and caspase 3 protein cleavage were prevented by adding the antioxidant, N-acetyl-cysteine (NAC), with decreased ROS production elicited by EPO1. Activation of ERK and JNK, but not p38, via phosphorylation induction was identified in EPO1- but not EPO- or EPO2-treated U87 and C6 cells, and this was blocked by adding NAC. However, the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125, showed no effect on EPO1-induced cytotoxicity in either cell type. Our findings demonstrate that 2,3-epoxide substitution significantly potentiates the apoptotic effect of vitamin K3 via stimulating ROS production, which may be useful in the chemotherapy of glioblastoma cells.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H₂DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

A novel role of catalase in detoxification of peroxynitrite in S. cerevisiae

The biological targets of peroxynitrite toxicity include wide array of biomolecules. Although several enzymes are found to be important components of cellular defense against peroxynitrite, the complete scenario is not totally understood. Yeast flavohemoglobin (YHB) and glutathione-dependent formaldehyde dehydrogenase (GS-FDH) confers resistance against nitric oxide and related reactive nitrogen species. In the present study, when subtoxic dose of peroxynitrite was applied to wild type, Δyhb1 and Δsfa1 strains of Saccharomyces cerevisiae, induction of cytosolic catalase was found at activity as well as gene expression level in mutants but not in wild type. Such induction was not due to intracellular reactive oxygen species (ROS) formation. Our in vitro studies confirmed the role of catalase in protection against peroxynitrite-mediated oxidation and nitration and also in peroxynitrite catabolism. This report is first of its kind regarding the novel role of catalase in peroxynitrite detoxification in Δyhb1 and Δsfa1 strains of S. cerevisiae.     

Interaction with Ppil3 leads to the cytoplasmic localization of Apoptin in tumor cells

Apoptin, a small protein encoded by chicken anemia virus (CAV), induces cell death specifically in cancer cells. In normal cells, Apoptin remains in the cytoplasm; whereas in cancerous cells, it migrates into the nucleus and kills the cell. Cellular localization appears to be crucial. Through a yeast two-hybrid screen, we identified human Peptidyl-prolyl isomerase-like 3 (Ppil3) as one of the Apoptin-associated proteins. Ppil3 could bind Apoptin directly, and held Apoptin in cytoplasm even in tumor cells. We then demonstrated that the nuclearcytoplasmic distribution of Apoptin is related to the expression level of intrinsic Ppil3. Moreover, extrinsic modifying of Ppil3 levels also resulted in nuclearcytoplasmic shuffling of Apoptin. The Apoptin P109A mutant, located between the putative nuclear localization and export signals, could significantly impair the function of Ppil3. Our results suggest a new direction for the localization mechanism study of Apoptin in cells.     

Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are among the important second messengers in abscisic acid (ABA) signaling in guard cells. In this study, to investigate specific roles of H₂O₂ in ABA signaling in guard cells, we examined the effects of mutations in the guard cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-aminotriazole (AT) on stomatal movement. The cat3 and cat1 cat3 mutations significantly reduced CAT activities, leading to higher basal level of H₂O₂ in guard cells, when assessed by 2′,7′-dichlorodihydrofluorescein, whereas they did not affect stomatal aperture size under non-stressed condition. In addition, AT-treatment at concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size, while basal H₂O₂ level increased extensively. In contrast, cat mutations and AT-treatment potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was observed in these mutants and wild type as well as in AT-treated guard cells. These results suggest that ABA-inducible cytosolic H₂O₂ elevation functions in ABA-induced stomatal closure, while constitutive increase of H₂O₂ do not cause stomatal closure.     

Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis through activation of JNK1 following engagement of membrane immunoglobulin

Engagement of membrane immunoglobulin (mIg) on WEHI-231 mouse B lymphoma cells results in growth arrest at the G1 phase of the cell cycle, followed by a reduction of mitochondrial membrane potential (ΔΨm) and apoptosis. WEHI-231 cells resemble immature B cells in terms of the cell surface phenotype and sensitivity to mIg engagement. However, the molecular mechanisms underlying mIg-induced loss of ΔΨm and apoptosis have not yet been established. In this study, we show that apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase 1 (JNK1) signaling pathway participates in mIg-induced apoptosis through the generation of reactive oxygen species (ROS). Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation immediately (5 min) induces hydrogen peroxide (H₂O₂) production with a substantial increase during later time points (36-48 h), accompanied by loss of ΔΨm and an increase in cells with sub-G1 DNA content. The anti-IgM-induced late-phase H₂O₂ production, loss of ΔΨm, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone, but enhanced substantially in cells overexpressing a constitutively active form of ASK1. These mIg-mediated events were also partially abrogated by ROS scavenger N-acetyl-l-cysteine (NAC). Taken together, these results suggest that mIg engagement induces H₂O₂ production leading to activation of ASK1-JNK1 pathway, creating a feedback amplification loop of ROS-ASK/JNK that leads to loss of ΔΨm and finally apoptosis.     

Omeprazole attenuates hyperoxic injury in H441 cells via the aryl hydrocarbon receptor

Hyperoxia contributes to the development of bronchopulmonary dysplasia in premature infants. Earlier we observed that aryl hydrocarbon receptor (AhR)-deficient mice are more susceptible to hyperoxic lung injury than AhR-sufficient mice, and this phenomenon was associated with a lack of expression of cytochrome P450 1A enzymes. Omeprazole, a proton pump inhibitor used in humans with gastric acid-related disorders, activates AhR in hepatocytes in vitro. However, the effects of omeprazole on AhR activation in the lungs and its impact on hyperoxia-induced reactive oxygen species (ROS) generation and inflammation are unknown. In this study, we tested the hypothesis that omeprazole attenuates hyperoxia-induced cytotoxicity, ROS generation, and expression of monocyte chemoattractant protein-1 (MCP-1) in human lung-derived H441 cells via AhR activation. Experimental groups included cells transfected with AhR small interfering RNA (siRNA). Hyperoxia resulted in significant increases in cytotoxicity, ROS generation, and MCP-1 production, which were significantly attenuated with the functional activation of AhR by omeprazole. The protective effects of omeprazole on cytotoxicity, ROS production, and MCP-1 production were lost in H441 cells whose AhR gene was silenced by AhR siRNA. These findings support the hypothesis that omeprazole protects against hyperoxic injury in vitro via AhR activation that is associated with decreased ROS generation and expression of MCP-1.     

Altered Ca signaling in cancer cells: Proto-oncogenes and tumor suppressors targeting IP3 receptors

Proto-oncogenes and tumor suppressors critically control cell-fate decisions like cell survival, adaptation and death. These processes are regulated by Ca^2 + signals arising from the endoplasmic reticulum, which at distinct sites is in close proximity to the mitochondria. These organelles are linked by different mechanisms, including Ca^2 +-transport mechanisms involving the inositol 1,4,5-trisphosphate receptor (IP₃R) and the voltage-dependent anion channel (VDAC). The amount of Ca^2 + transfer from the endoplasmic reticulum to mitochondria determines the susceptibility of cells to apoptotic stimuli. Suppressing the transfer of Ca^2 + from the endoplasmic reticulum to the mitochondria increases the apoptotic resistance of cells and may decrease the cellular responsiveness to apoptotic signaling in response to cellular damage or alterations. This can result in the survival, growth and proliferation of cells with oncogenic features. Clearly, proper maintenance of endoplasmic reticulum Ca^2 + homeostasis and dynamics including its links with the mitochondrial network is essential to detect and eliminate altered cells with oncogenic features through the apoptotic pathway. Proto-oncogenes and tumor suppressors exploit the central role of Ca^2 + signaling by targeting the IP₃R. There are an increasing number of reports showing that activation of proto-oncogenes or inactivation of tumor suppressors directly affects IP₃R function and endoplasmic reticulum Ca^2 + homeostasis, thereby decreasing mitochondrial Ca^2 + uptake and mitochondrial outer membrane permeabilization. In this review, we provide an overview of the current knowledge on the proto-oncogenes and tumor suppressors identified as IP₃R-regulatory proteins and how they affect endoplasmic reticulum Ca^2 + homeostasis and dynamics.     

Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide

Background

Hydrogen peroxide (H₂O₂) is an important signaling compound that has recently been identified as a new substrate for several members of the aquaporin superfamily in various organisms. Evidence is emerging about the physiological significance of aquaporin-facilitated H₂O₂ diffusion.

Scope of review

This review summarizes current knowledge about aquaporin-facilitated H₂O₂ diffusion across cellular membranes. It focuses on physicochemical and experimental evidence demonstrating the involvement of aquaporins in the transport of this redox signaling compound and discusses the regulation and structural prerequisites of these channels to transmit this signal. It also provides perspectives about the potential importance of aquaporin-facilitated H₂O₂ diffusion processes and places this knowledge in the context of the current understanding of transmembrane redox signaling processes.

Major conclusions

Specific aquaporin isoforms facilitate the passive diffusion of H₂O₂ across biological membranes and control H₂O₂ membrane permeability and signaling in living organisms.

General significance

Redox signaling is a very important process regulating the physiology of cells and organisms in a similar way to the well-characterized hormonal and calcium signaling pathways. Efficient transmembrane diffusion of H₂O₂, a key molecule in the redox signaling network, requires aquaporins and makes these channels important players in this signaling process. Channel-mediated membrane transport allows the fine adjustment of H₂O₂ levels in the cytoplasm, intracellular organelles, the apoplast, and the extracellular space, which are essential for it to function as a signal molecule. This article is part of a Special Issue entitled Aquaporins.     

PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3

Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy.