Dose- and time-dependent effects of doxorubicin on cytotoxicity, cell cycle and apoptotic cell death in human colon cancer cells.

The cytostatic drug doxorubicin is a well-known chemotherapeutic agent which is used in treatment of a wide variety of cancers. A key factor in the response of cancer cells to chemotherapeutic drugs is the activation of the apoptotic pathway, a pathway that is often impaired in chemoresistant colon cancer cells. The aim of the present study was to investigate the effects of doxorubicin in Hct-116 human colon carcinoma cells in order to clarify if a time/concentration range for optimal doxorubicin-induced apoptosis exists. We compared a treatment schedule were cells were bolus incubated for 3h with doxorubicin followed by 24h in drug-free medium, with a continuous doxorubicin treatment schedule for 24h. Bolus incubation was carried out to determine effects of doxorubicin accumulated during the first 3h, whereas continuous incubation allowed further (continuous) exposure to doxorubicin. We found that bolus (3h) treatment with doxorubicin resulted in a dose-dependent decrease of viable cells and concomitant increase of apoptosis. Additionally, bolus (3h) doxorubicin incubation led to phosphorylation of p53 at serine 392, induction of p21, G2 arrest and increase of proapoptotic protein Bax. In contrast, continuous (24h) treatment with doxorubicin reduced the number of living cells with no parallel raise in the amount of dead cells. Continuous (24h) treatment with 5 microM doxorubicin resulted in cell cycle arrest in G0/G1 phase that was neither accompanied by phosphorylation and activation of p53 nor enhanced expression of p21. These results suggest that doxorubicin is able to induce cell death by apoptosis only at particular dose and treatment conditions and imply a completely different cellular response following bolus or continuous exposure to doxorubicin.

The use of Fluoro-Jade in primary neuronal cell cultures.

Fluoro-Jade (FJ) and its derivatives are widely used for histological staining of neurons undergoing neurodegeneration. With this dye, the entire structure of these neurons can be stained in a fast and reliable way in histopathological slices of the brain, with results comparable to those obtained with other methods such as the Nissle technique or silver staining. The question arose as to whether this method might be useful for in vitro neuronal cell cultures. Primary cortical neuronal cell cultures have been used as a sensitive and reliable system to detect compounds which induce neurodegenerative lesions (Schmuck et al. 2000). Additionally, various biochemical endpoints in this system allow the mode of action of these compounds to be identified. The target mechanism of FJ staining is unknown, and it may therefore be useful to compare FJ staining with one of the central endpoints in compound-induced neurodegeneration, interaction with the cytoskeleton as demonstrated by accumulations of neurofilaments (200 kD). Cortical neuronal cells were cultivated under standardized serum-free conditions. Once they had developed a stable network, the cells were treated with acrylamide, mipafox, diethyldithiocarbamate, glutamate, paraquat, paraoxon, and IDPN (ss,ss-imino dipropionitrile) for 7 days in the concentration range of 0.1-50 microg/ml. One half of the cell culture samples were tested directly after 7 days, the others were allowed to recover during a 7-day treatment-free period. Subsequently viability testing and quantification for FJ staining were performed. All compounds except paraoxon increased FJ staining after 7 days, and this signal increased slightly during the recovery period with glutamate and acrylamide. With mipafox and IDPN the signal decreased slightly. Paraoxon increased FJ staining only after the recovery period. The intensity of FJ staining did not always correlate with neurofilament destruction or cytotoxicity. It can therefore be assumed that FJ targets a different cellular endpoint. Interestingly, paraoxon, a compound which does not induce neurodegeneration, increased FJ staining only in the recovery phase; this pointed to a neurotoxic mechanism which sets it apart from the other model compounds.

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-alpha induced NF-kappaB activation and sensitizes MCF-7 cells against TNF-alpha.

The pleiotropic cytokine tumor necrosis factor alpha (TNF-alpha) can induce apoptosis but also supports cell survival pathways. Among the possible anti-apoptotic mechanisms of TNF-alpha is the activation of the transcription factor NF-kappaB. Since reactive oxygen species (ROS) are assumed to contribute to TNF-alpha mediated cytotoxicity but can also facilitate NF-kappaB activation this study investigates the relationship between TNF-alpha treatment, NF-kappaB activation and the expression of the anti-oxidative enzyme catalase. TNF-alpha 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 H(2)O(2), caused a transient nuclear p65 translocation upon TNF-alpha treatment as compared to the sustained NF-kappaB activation in wild type cells. This was due to a lack of sufficient H(2)O(2) to co-stimulate NF-kappaB activation as demonstrated by the observation that addition of exogenous H(2)O(2) led to a second increase of NF-kappaB activity. The rapid decline of nuclear translocation of NF-kappaB in the catalase overexpressing cells resulted in a slower increase of NF-kappaB mediated reporter gene expression. These results indicate that TNF-alpha mediated downregulation of catalase expression and accordingly sufficient H(2)O(2) is required for appropriate function of the NF-kappaB dependent survival pathway.

Increased expression of catalase in human hepatoma cells by the soy isoflavone, daidzein.

The reduced incidence of cancer that has been observed in Asian population traditionally consuming soy-based food has been linked to the antioxidant potential of soy isoflavones, in particular daidzein and genistein. The present study was undertaken in order to test the antioxidative potential of daidzein and to examine the effect of daidzein treatment on the expression of the antioxidant enzyme catalase in the human hepatoma cell lines Huh-7 and HepG2. Daidzein itself did not display radical scavenging activity but it significantly increased the activity of the antioxidant enzyme catalase. Huh-7 cells were much more susceptible to daidzein cytotoxicity than HepG2 cells and showed much lower basal activity in luciferase reporter gene assays with the 3.2 kb fragment of the human catalase promoter. However, treatment with daidzein at a non-toxic concentration resulted in a similar induction of promoter activity in both cell lines. Reporter gene studies with different promoter constructs in HepG2 cells restrict the potential localization of the main regulatory elements for basal and inducible activity of the catalase promoter to a region approximately 120 bp to 300 bp upstream of the start codon of the catalase gene. From our results, we conclude that in human hepatoma cells daidzein at a non-toxic concentration increases the activity of human catalase and induces the transcription of the catalase gene via interaction with the proximal part of the promoter.

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).

Investigations of protective effects of the flavonoids quercetin and rutin on stress resistance in the model organism Caenorhabditis elegans.

Oxidative stress as a result of excessive generation of reactive oxygen species (ROS) or diminished antioxidative defence is regarded as a main factor in the pathophysiology of various diseases and ageing. Many flavonoids that are present in herbal edibles have antioxidative properties and possess a remarkable spectrum of biochemical and pharmacological actions. They are assumed to exert beneficial effects but the precise biological mechanism of their action is unknown. In this project, we studied effects of the flavonoids quercetin and rutin in the multicellular model organism Caenorhabditis elegans that exhibits a strong conservation in molecular and cellular pathways to mammals. Both flavonoids reduced the ROS accumulation at thermal stress and the extent of induced oxidative stress with quercetin having a stronger impact. The higher antioxidative activity of quercetin may explain the protection against lethal thermal stress and the reduction in accumulation of the ageing marker lipofuscin exerted by quercetin but not by rutin. The subcellular distribution of the FoxO transcription factor DAF-16 was only affected by quercetin indicating a modulatory effect of quercetin on signalling cascade(s). These results suggest that quercetin may act as an antioxidant as well as a modulator of cellular signalling processes to exert its protective properties.

Downregulation of NF-kappaB activation in a H4IIE transfectant insensitive to doxorubicin-induced apoptosis.

Cytostatic drugs are administered to cancer patients in order to drive the tumor cells into apoptosis by DNA damage signalling pathway(s). DNA damage also leads to NF-kappaB activation, and it is controversial whether this is exclusively part of a survival process, thus enabling drug resistance, or whether it can also lead to a pro-apoptotic response, thus supporting the therapeutic purpose of drug administration. In the present work, the pathway and outcome of NF-kappaB activation was compared in the doxorubicin sensitive H4IIE rat hepatoma cell and the H4IIE-derived transfectant Yv2-12 which is insensitive to doxorubicin induced apoptosis. In the wild type H4IIE cell, doxorubicin induces serine 536 phosphorylation and nuclear translocation of p65 which however results in reduced rather than increased expression of the anti-apoptotic protein XIAP. Apoptosis in H4IIE cells is accompanied by rapid production of intracellular reactive oxygen species, caspase activation and increased expression of the pro-apoptotic protein Bax. The doxorubicin-insensitive Yv2-12 transfectant differs from its wild type counterpart by the complete failure to activate NF-kappaB in response to doxorubicin. In contrast, serine 536 phosphorylation and nuclear translocation of p65 are even reduced by doxorubicin treatment while the expression of XIAP and Bax remain virtually unchanged. These results show that NF-kappaB activation by doxorubicin in our experimental system proceeds by an atypical pathway resulting in a pro-apoptotic effect and that insensitivity to doxorubicin-induced apoptosis was accompanied by a loss of NF-kappaB activation.

Protective and detrimental effects of kaempferol in rat H4IIE cells: implication of oxidative stress and apoptosis.

Flavonoids are ubiquitous substances in fruits and vegetables. Among them, the flavonol kaempferol contributes up to 30% of total dietary flavonoid intake. Flavonoids are assumed to exert beneficial effects on human health, e.g., anticancer properties. For this reason, they are used in food supplements at high doses. The aim of this project was to determine the effects of kaempferol on oxidative stress and apoptosis in H4IIE rat hepatoma cells over a broad concentration range. Kaempferol is rapidly taken up and glucuronidated by H4IIE cells. The results demonstrate that kaempferol protects against H2O2-induced cellular damage at concentrations which lead to cell death and DNA strand breaks in the absence of H2O2-mediated oxidative stress. Preincubation with 50 microM kaempferol exerts protection against the loss of cell viability induced by 500 microM H2O2 (2 h) while the same concentration of kaempferol reduces cell viability by 50% in the absence of H2O2 (24 h). Preincubation with 50 microM kaempferol ameliorates the strong DNA damage induced by 500 microM H2O2 while 50 microM kaempferol leads to a significant increase of DNA breakage in the absence of H2O2. Preincubation with 50 microM kaempferol reduces H2O2-mediated caspase-3 activity by 40% (4 h) while the same concentration of kaempferol leads to the formation of a DNA ladder in the absence of H2O2 (24 h). It is concluded that the intake of high dose kaempferol in food supplements may not be advisable because in our cellular model protective kaempferol concentrations can also induce DNA damage and apoptosis by themselves.