Andreas Kampkötter

A stress-responsive glutathione S-transferase confers resistance to oxidative stress in Caenorhabditis elegans

Previous studies demonstrated that the Caenorhabditis elegans GST-p24 is upregulated at the steady state mRNA level in response to oxidative stress. A transcriptional upregulation was confirmed in the current study by analyzing Ce-GST-p24 promoter-reporter constructs in transgenic C. elegans strains CL2166 and CL3166. The transgenic strain BL1, which overexpresses the Ce-GST-p24 enzyme (as a GFP fusion protein controlled by its own promoter), was generated to investigate the function of this enzyme in vivo. Stress experiments with BL1 demonstrated an increased resistance to intracellularly induced oxidative stress, as compared to wild type. The consequences of a decrease in the Ce-GST-p24 enzyme concentration were examined by RNAi-treatment of BL1 C. elegans to silence both the endogene and the transgene Ce-GST-p24 and by the analysis of the K08F4.7 homozygous deletion mutant. In both cases, the reduced Ce-GST-p24 enzyme level resulted in a significant decrease in the stress resistance of the nematodes. These results clearly demonstrate a direct correlation between the concentration of Ce-GST-p24 and the resistance to oxidative stress. We have demonstrated for the first time that manipulation of the expression of a single GST can modulate the organismal response to oxidative stress. The enzymatic activity of this detoxification enzyme was examined with various substrates, giving emphasis to lipid peroxidation products. The Ce-GST-p24 was also localized in BL1 C. elegans by confocal laser-scanning microscopy, revealing a wide-spread distribution profile.

Effects of the flavonoids kaempferol and fisetin on thermotolerance, oxidative stress and FoxO transcription factor DAF-16 in the model organism Caenorhabditis elegans

Flavonoids present in many herbal edibles possess a remarkable spectrum of biochemical and pharmacological actions and they are assumed to exert beneficial effects to human health. Although the precise biological mechanisms of their action has not been elucidated yet many of the protective properties of flavonoids are attributed to their antioxidative activity since oxidative stress is regarded as a main factor in the pathophysiology of various diseases and ageing. Oxidative stress results from excessive generation of reactive oxygen species (ROS) or diminished antioxidative defence and thus antioxidants are able to counteract such situations. We used the multicellular model organism Caenorhabditis elegans that is conserved in molecular and cellular pathways to mammals to examine the effects of the flavonoids kaempferol and fisetin with respect to their protective action in individual living worms. Both flavonoids increased the survival of C. elegans, reduced the intracellular ROS accumulation at lethal thermal stress, and diminished the extent of induced oxidative stress with kaempferol having a stronger impact. Kaempferol but not fisetin attenuated the accumulation of the ageing marker lipofuscin suggesting a life prolonging activity of this flavonoid. In addition to these effects that may be attributed to their antioxidative potential kaempferol and fisetin caused a translocation of the C. elegans FoxO transcription factor DAF-16 from the cytosol to the nucleus indicating a modulatory influence of both flavonoids on signalling cascade(s).

Enniatins A1, B and B1 from an endophytic strain of Fusarium tricinctum induce apoptotic cell death in H4IIE hepatoma cells accompanied by inhibition of ERK phosphorylation.

Enniatins are mycotoxins which have important impact on human health, e.g. as contaminants of cereals, but also are discussed as possible anticancer agents. We investigated toxic effects of enniatins A1, B and B1 isolated from Fusarium tricinctum on different cancer cell lines. The enniatins showed moderate activity in HepG2 and C6 cells (EC(50)-values approximately 10-25 microM), but were highly toxic in H4IIE cells (EC(50)-values approximately 1-2.5 microM). In H4IIE cells, all enniatins increased caspase 3/7 activity and nuclear fragmentation as markers for apoptotic cell death. Enniatin A1, enniatin B1, and, to a lesser extent, also enniatin B decreased the activation of extracellular regulated protein kinase (ERK) (p44/p42), a mitogen-activated protein kinase which is associated with cell proliferation. Furthermore, enniatins A1 and B1, but not enniatin B were able to inhibit moderately tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation. Screening of 24 additional protein kinases involved in signal transduction pathways (cell proliferation, survival, angiogenesis and metastasis) showed no inhibitory activity of enniatins. We conclude that enniatins A1 and B1 and, to a lesser extent, enniatin B may possess anticarcinogenic properties by induction of apoptosis and disruption of ERK signalling pathway. Further analysis of these substances is necessary to analyse their usefulness for cancer therapy.

Antioxidant enzymes and apoptosis.

The role of antioxidant enzymes can be interpreted in terms of fine tuning of the concentration of reactive oxygen species which are required in the redox regulation of the cell cycle and of programmed cell death. This review summarizes findings from papers published in the last few years which deal with the relation between apoptosis and the two antioxidant enzymes, manganous superoxide dismutase (MnSOD) and catalase. With respect to MnSOD, the literature is much in favor of an inhibitory action in apoptosis. Increased MnSOD activity has been shown to prevent cell death via the receptor‐mediated apoptotic pathway as well as cell death via the mitochondrial pathway. The literature on the influence of catalase activity on apoptosis is less consistent. Evidence for both an antiapoptotic and a proapoptotic role of catalase can be found. From the results reviewed here, two schemes for the involvement of MnSOD and catalase in the regulation of apoptosis can be extracted: 1) Both MnSOD and catalase inhibit apoptosis by removing superoxide anion radicals or H2O2, respectively, because these reactive oxygen species are mediators required for the apoptotic program or inhibit a survival pathway. 2) An increase in H2O2 by downregulation or inhibition of catalase activity and/or downregulation of MnSOD activity inhibits apoptosis while a decrease in H2O2 by upregulation of catalase activity and/or upregulation of MnSOD activity supports apoptosis, possibly because of a supportive role of H2O2 in a survival pathway. The data reported so far do not allow for an explanation why some cell models appear to fit the first scheme while the second scheme appears to correctly describe other cell models. The present state of the literature reveals that antioxidant enzymes play a more intricate role in cell physiology than previously assumed.

Functional analysis of the glutathione S-transferase 3 from Onchocerca volvulus (Ov-GST-3): A parasite GST confers increased resistance to oxidative stress in Caenorhabditis elegans

This study examined the genomic organisation of the coding region of the glutathione S-transferase 3 (Ov-GST-3) from the human parasitic nematode Onchocerca volvulus; alternative splicing leads to three different transcripts (Ov-GST-3/1; Ov-GST-3/2 and Ov-GST-3/3). Since the expression of Ov-GST-3 is inducible by oxidative stress, it is assumed that it is involved in the defense against reactive oxygen species (ROS) resulting from cellular metabolism. Furthermore, we suggest that Ov-GST-3 plays an important role in the protection of the parasite against ROS derived from the hosts immune system. To experimentally investigate these speculations, we generated Caenorhabditis elegans lines transgenic for Ov-GST-3 (AK1) and examined their resistance to artificially generated ROS. The AK1 worms (extrachromosomal and integrated lines) were found to be much more resistant to internal (juglone) and external (hypoxanthine/xanthine oxidase) oxidative stress than wild-type C.elegans worms. RNA interference experiments targeted to the Ov-GST-3 transcripts resulted in decreased resistance, confirming that this effect is due to the transgenic expression of Ov-GST-3. These results clearly demonstrate that the Ov-GST-3 gene confers an increased resistance to oxidative stress. This study also shows the applicability of C.elegans as a model organism for the functional characterization of genes from (parasitic) nematode species which are not accessible to genetic manipulations.

The forkhead transcription factor FOXO4 sensitizes cancer cells to doxorubicin-mediated cytotoxicity

The forkhead superfamily of transcription factors, which play major roles in control of cellular proliferation, oxidative stress and apoptosis, are becoming more and more considered as crucial therapeutic targets in cancer. In this study, we addressed the contribution of class O of forkhead box transcription factor (FOXO) 4 transcription factor, a forkhead superfamily member, to cytotoxicity mediated by the anthracyclic drug doxorubicin. FOXO4 can be phosphorylated by phosphatidylinositol-3-kinase/AKT signaling resulting in its inactivation and nuclear exclusion. Under stress conditions, FOXO4 can be phosphorylated via jun N-terminal kinase (JNK) leading to increased transcriptional activation of the transcription factor. Our results show that doxorubicin incubation led to phosphorylation of AKT and concomitantly to AKT-dependent inactivation and nuclear exclusion of the tumor suppressor FOXO4 in Hct-116 cells. We found that inhibition of FOXO4 nuclear exclusion by blockage of AKT phosphorylation following overexpression of dominant-negative AKT enhanced doxorubicin-mediated cytotoxicity. Overexpression of wild-type FOXO4 led to an increase in doxorubicin-mediated cytotoxicity, which was further exacerbated by overexpression of a solely nuclear-localized FOXO4 mutant. In contrast, though doxorubicin resulted in JNK activation, modulation of JNK-dependent regulation of FOXO4 was of no effect to doxorubicin cytotoxicity. These results show for the first time that in Hct-116 cells sustained nuclear localization of FOXO4 seems to be one crucial point enhancing doxorubicin-induced cytotoxicity and apoptosis. Targeting FOXO4 or AKT may lead to new chances in sensitizing cancer cells to cytostatic drugs thereby allowing use of lower drug concentrations and minimizing drug-induced adverse effects in patients.

Catalase overexpression impairs TNF-α induced NF-κB activation and sensitizes MCF-7 cells against TNF-α

The pleiotropic cytokine tumor necrosis factor alpha (TNF‐α) can induce apoptosis but also supports cell survival pathways. Among the possible anti‐apoptotic mechanisms of TNF‐α is the activation of the transcription factor NF‐κB. Since reactive oxygen species (ROS) are assumed to contribute to TNF‐α mediated cytotoxicity but can also facilitate NF‐κB activation this study investigates the relationship between TNF‐α treatment, NF‐κB activation and the expression of the anti‐oxidative enzyme catalase. TNF‐α treatment caused downregulation of catalase expression in MCF‐7, Caco‐2 and Hct‐116 cancer cell lines. Overexpression of catalase in MCF‐7 cells, resulting in lower intracellular ROS levels upon challenge with H2O2, caused a transient nuclear p65 translocation upon TNF‐α treatment as compared to the sustained NF‐κB activation in wild type cells. This was due to a lack of sufficient H2O2 to co‐stimulate NF‐κB activation as demonstrated by the observation that addition of exogenous H2O2 led to a second increase of NF‐κB activity. The rapid decline of nuclear translocation of NF‐κB in the catalase overexpressing cells resulted in a slower increase of NF‐κB mediated reporter gene expression. These results indicate that TNF‐α mediated downregulation of catalase expression and accordingly sufficient H2O2 is required for appropriate function of the NF‐κB dependent survival pathway. J. Cell. Biochem. 103: 1497–1511, 2008.

Hexa-, hepta- and nonaprenylhydroquinones isolated from marine sponges Sarcotragus muscarum and Ircinia fasciculata inhibit NF-k B signalling in H4IIE cells

Objectives Marine organisms have proven to be a rich source of potent pharmacologically active compounds. Three polyprenyl‐1,4‐hydroquinone derivates (hexaprenyl‐1,4‐hydroquinone, heptaprenyl‐1,4‐hydroquinone and nonaprenyl‐1,4‐hydroquinone) were isolated from the Zoobenthos‐inhabiting sponges Sarcotragus muscarum and Ircinia fasciculata from the Eastern Mediterranean Sea (phylum: Porifera; class: Demospongiae).