An AMP Kinase-pathway dependent integrated stress response regulates ageing and longevity.

The purpose of this article is to investigate the role of the AMP-kinase pathway (AMPK pathway) in the induction of a concomitant set of health benefits by exercise, numerous drugs, and health ingredients, all of which are adversely affected by ageing. Despite the AMPK pathway being frequently mentioned in relation to both these health effects and ageing, it appears challenging to understand how the activation of a single biochemical pathway by various treatments can produce such a diverse range of concurrent health benefits, involving so many organs. We discovered that the AMPK pathway functions as an integrated stress response system because of the presence of a feedback loop in it. This evolutionary conserved stress response system detects changes in AMP/ATP and NAD/NADH ratios, as well as the presence of potential toxins, and responds by activating a common protective transcriptional response that protects against aging and promotes longevity. The inactivation of the AMPK pathway with age most likely explains why ageing has a negative impact on the above-mentioned set of health benefits. We conclude that the presence of a feedback loop in the AMP-kinase pathway positions this pathway as an AMPK-ISR (AMP Kinase-dependent integrated stress response) system that responds to almost any type of (moderate) environmental stress by inducing various age-related health benefits and longevity.

Toxicological evaluation of pomegranate seed oil.

In this manuscript, the toxicology and safety of pomegranate seed oil (PSO) was evaluated by in vitro (Ames, chromosomal aberration), and in vivo toxicity tests (acute toxicity and 28-day toxicity in Wistar rats). No mutagenicity of PSO was observed in the absence and presence of metabolic activation up to precipitating concentrations of 5000 microg/plate (Ames test) or 333 microg/ml (chromosome aberration test). The acute oral toxicity study revealed no significant findings at 2000 mg PSO/kg body weight. In the 28-day dietary toxicity study PSO was dosed at concentrations of 0, 10,000, 50,000 and 150,000 ppm, which resulted in a mean intake of 0-0, 825-847, 4269-4330 and 13,710-14,214 mg PSO/kg body weight per day in males-females, respectively. At 150,000 ppm dietary exposure to PSO, a much higher dose than the level of PSO that elicits antidiabetic and anti-inflammatory efficacy, increased hepatic enzyme activities determined in plasma (aspartate, alanine aminotransferase and alkaline phosphatase) and increased liver-to-body weight ratios were observed. However, these effects might be the result of a physiological response to exposure to a very high level of a fatty acid which is not part of the normal diet, and are most likely not toxicologically relevant. The no observable adverse effect level (NOAEL) was 50,000 ppm PSO (=4.3 g PSO/kg body weight/day).

Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells.

Doxorubicin accumulation defects in multidrug resistant tumour cells are generally small in comparison to the resistance factors. Therefore additional mechanisms must be operative. In this paper we show by a quantitative approach that doxorubicin resistance in several P-glycoprotein-positive non-small cell lung cancer and breast cancer multidrug resistant cell lines can be explained by a summation of accumulation defect and alterations in the efficacy of the drug once present in the cell. This alteration of efficacy was partly due to changes in intracellular drug localisation, characterised by decreased nuclear/cytoplasmic doxorubicin fluorescence ratios (N/C-ratios). N/C-ratios were 2.8-3.6 in sensitive cells, 0.1-0.4 in cells with high (> 70-fold) levels of doxorubicin resistance and 1.2 and 1.9 in cells with low or intermediate (7.5 and 24-fold, respectively) levels of doxorubicin resistance. The change of drug efficacy was reflected by an increase in the total amount of doxorubicin present in the cell at equitoxic (IC50) concentrations. N/C ratios in highly resistant P-glycoprotein-containing cells could be increased with the resistance modifier verapamil to values of 1.3-2.7, a process that was paralleled by a decrease of the cellular doxorubicin amounts present at IC50. At the low to moderate residual levels of resistance, obtained with different concentrations of verapamil, a linear relationship between IC50 and cellular doxorubicin amounts determined at IC50 was found. This shows that at this stage of residual resistance, extra reversal by verapamil should be explained by further increase of drug efficacy rather than by increase of cellular drug accumulation. A similar relationship was found for P-glycoprotein-negative MDR cells with low levels of resistance. Since in these cells N/C ratios could not be altered, verapamil-induced decrease of IC50 must be due to increased drug efficacy by action on as yet unidentified targets. Although the IC50 of sensitive human cells cannot be reached with resistance modifiers, when using these relationships it can be shown by extrapolation that cellular and nuclear doxorubicin amounts at IC50 at complete reversal of resistance were the same as in sensitive cells. It is concluded that doxorubicin resistance factors for multidrug resistant cells can for a large part, and in the case of P-glycoprotein-containing cells probably fully, be accounted for by decreased amounts of drug at nuclear targets, which in turn is characterised by two processes only: decreased cellular accumulation and a shift in the ratio nuclear drug/cytoplasmic drug.

Doxorubicin (adriamycin): a critical review of free radical-dependent mechanisms of cytotoxicity.

The antineoplastic drug doxorubicin is capable of generating a variety of free radical species in subcellular systems and this capacity has been considered critical for its antitumor action. However, for most tumor cell lines this mechanism of cytotoxicity does not appear to play a major role. Free radical-independent cytotoxicity mechanisms, taking place in the nuclear compartment of the cell, may more likely be involved in the antitumor effect of doxorubicin.

Effect of artificial electron acceptors on the cytotoxicity of mitomycin C and doxorubicin in human lung tumor cells.

The cytotoxicities of mitomycin C (MMC) and doxorubicin (DOX) have been proposed to depend on intracellular reduction by reduced flavoproteins. We investigated whether MMC- and DOX-induced cytotoxicity could be inhibited by competing for electrons from reduced flavoproteins by the artificial electron acceptors phenazine methosulfate (PMS), menadione (MEN) and methylene blue (MB). In intact SW-1573 human lung tumor cells these compounds proved to be excellent electron acceptors, as judged from their capacity to induce high levels of cyanide-resistant respiration. In addition, PMS, MEN and MB were found to decrease the cytotoxicity of MMC, by 90, 63 and 29%, respectively, at concentrations that were themselves completely nontoxic. In contrast, DOX cytotoxicity was not detectably affected. These results suggest that in SW-1573 cells flavoprotein-mediated bioreduction is required for the cytotoxic effect of MMC, but not for that of DOX.

Correlation of multidrug resistance with decreased drug accumulation, altered subcellular drug distribution, and increased P-glycoprotein expression in cultured SW-1573 human lung tumor cells.

Four multidrug-resistant variants of the human squamous lung cancer cell line SW-1573 with levels of doxorubicin resistance ranging from 10- to 2000-fold were characterized with respect to drug accumulation and efflux, subcellular drug distribution pattern, antioxidant defenses, and P-glycoprotein expression. For all these parameters except the antioxidant defenses a correlation was observed with the level of doxorubicin resistance; with increasing drug resistance cellular drug accumulation capacity (as measured for doxorubicin) progressively decreased, initial drug efflux rates (as measured for daunorubicin) progressively increased, while the subcellular doxorubicin distribution (as measured by fluorescence microscopy) gradually shifted from a "mainly nuclear" to a "mainly cytoplasmic" pattern. Our data suggest that in the present set of cell lines the same mechanism of resistance is operating at all levels of doxorubicin resistance.

Increased cytosolic pH in multidrug-resistant human lung tumor cells: effect of verapamil.

In a set of four increasingly multidrug-resistant variants of SW-1573 human lung tumor cells, the pHi (i.e., steady-state cytosolic pH) increased up to 0.44 U as a function of the level of doxorubicin resistance. The elevated pHi in the most resistant (2,000-fold) variant dropped to the control level upon addition of verapamil, a known inhibitor of P-glycoprotein activity. These data suggest that, in the absence of xenobiotic substrates, P-glycoprotein activity can affect cellular pHi. This finding may be important for the elucidation of the physiological function of this protein.

Immunohistochemical detection of P-glycoprotein in human tumor cells with a low degree of drug resistance.

We report the immunohistochemical detection of the 170-180 kDa multi-drug-resistance-related P-glycoprotein in human tumor cells with a low level of resistance. A series of human squamous lung cancer cell lines with increasing levels of resistance to doxorubicin (DOX) was developed and stained for P-glycoprotein, using the JSB-IMAb. Subline SW1573/50A with a 4- to 6-fold cross-resistance to daunorubicin (DNR) and vincristine (VCR) showed rather uniform positive staining for P-glycoprotein apparently at cytoplasmic sites. Only in cells with higher degrees of resistance (greater than 10-fold) could plasma-membrane-associated P-glycoprotein be made visible. DNR efflux was increased in SW1573/50A as compared to the parent line SW1573 (52 and 70% DNR were retained during 3 min efflux respectively). Verapamil partially reversed DNR and VCR resistance in SW1573/50A. Cells obtained from a metastasized renal cell carcinoma and cultured in vitro stained in a similar way to SW1573/50A and showed some sensitivity to verapamil modulation of VCR cytotoxicity. Our results suggest that weakly resistant cancer cells obtained from patients can be routinely detected with JSB-I on cytospins, and implicate that in such weakly resistant cells P-glycoprotein may be present, while plasma membrane expression is not yet readily detectable.

Effect of endogenous glutathione, superoxide dismutases, catalase, and glutathione peroxidase on adriamycin tolerance of Chinese hamster ovary cells.

Based on the concept that activated oxygen species are causally involved in Adriamycin toxicity, endogenous antioxidant defenses are expected to be important determinants of cellular Adriamycin tolerance. We have tested this prediction by making use of an oxygen-resistant variant subline of Chinese hamster ovary cells (CHOr), which is characterized by increased levels of glutathione, copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase. The levels of antioxidant defenses in wild-type CHO (CHOs) cells were within the range reported for human tumor cell lines, except for catalase, which was comparatively high. Oxygen-tolerant CHOr cells, which contained 4.3-fold more catalase activity than CHOs cells, were proportionally more resistant to H2O2, indicating that catalase activity in wild-type CHOs cells was still limiting H2O2 tolerance. The Adriamycin sensitivity of CHOs cells was compared to that of CHOr cells by clonogenic cell survival. After correcting for differential drug uptake in CHOs and CHOr cells, no significant difference in Adriamycin sensitivity could be detected. Furthermore, drug-induced cyanide-resistant oxygen consumption and electron spin resonance data indicated that both cell strains were equally efficient in reducing Adriamycin to its semiquinone radical and in generating activated oxygen species through oxidation-reduction cycling. These results indicate that Adriamycin tolerance of wild-type CHO cells, as determined by clonogenic cell survival, is not limited by endogenous glutathione, copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, or glutathione peroxidase.

Transport of trehalose in Salmonella typhimurium.

We have studied trehalose uptake in Salmonella typhimurium and the possible involvement of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in this process. Two transport systems could recognize and transport trehalose, the mannose PTS and the galactose permease. Uptake of trehalose via the latter system required that it be expressed constitutively (due to a galR or galC mutation). Introduction of a ptsM mutation, resulting in a defective IIMan/IIIMan system, in S. typhimurium strains that grew on trehalose abolished growth on trehalose. A ptsG mutation, eliminating IIGlc of the glucose PTS, had no effect. In contrast, a crr mutation that resulted in the absence of IIIGlc of the glucose PTS prevented growth on trehalose. The inability of crr and also cya mutants to grow on trehalose was due to lowered intracellular cyclic AMP synthesis, since addition of extracellular cyclic AMP restored growth. Subsequent trehalose metabolism could be via a trehalose phosphate hydrolase, if trehalose phosphate was formed via the PTS, or trehalase. Trehalose-grown cells contained trehalase activity, but we could not detect phosphoenolpyruvate-dependent phosphorylation of trehalose in toluenized cells.

Antigen-presenting cell function of dendritic cells and macrophages in proliferative T cell responses to soluble and particulate antigens.

The capacity of dendritic cells (DC) and macrophages (M phi) to present soluble and particulate antigen was tested in an ovalbumin (OVA)-specific T cell proliferation assay. In a comparative investigation we found that both DC and M phi were able to present soluble OVA, but that only M phi could present insolubilized OVA to T cells. DC were found to be able to present OVA in collaboration with M phi. The failure of DC to present insolubilized OVA is probably caused by their inability to endocytose these antigens. DC appeared not to endocytose substantial amounts of soluble OVA either. In contrast to M phi, antigen presentation by DC is not blocked by lysosomotropic drugs. Taken together, these observations suggest that DC can present soluble protein antigens without intracellular degradation.

Dendritic cells as accessory cells in antigen-specific murine T lymphocyte proliferation in vitro.

The role of dendritic cells in antigen-induced murine T lymphocyte activation was studied by addition of purified dendritic cells to purified lymph node T lymphocytes from ovalbumin-primed mice. In the presence of the priming antigen T cells generated an antigen-specific response. The response was at least 3-fold higher with the use of a modified IMDM culture medium. The complete requirement for accessory cells was demonstrated only when nylon wool-purified T lymphocytes were thoroughly depleted of Ia antigen-expressing cells. Dendritic cells as well as peritoneal exudate macrophages were equally effective as antigen-presenting cells.