Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anti-cancer therapy. We previously reported that a combination of TNF and CHX efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. While ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX.Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, while its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.

In a Wistar rat model, prolonged supplementation of mustard seed (MS) to the diet significantly ameliorates the induction of colorectal carcinomas by 1,2-dimethylhydrazine (DMH). The expression of the splenocyte major histocompatibility complex class I (MHCI) was found significantly enhanced, whereas that of the major histocompatibility complex class II (MHCII) was significantly decreased. Compared to that of control animals, the proportion of spleenic B- and dendritic cells (DC) was amplified in the MS group. The expressions of MHCI, as well as that of MHCII, were increased in DC cells; whereas in B cells, MHCI expression was augmented but that of MHCII moderately decreased. The percentages of CD8+CD28+ and CD4+CD28+ cells were increased in the MS group, while the CD4+CD25+Foxp3+ subset was depressed. Plasma analysis showed that DMH-exposure induced amplified amounts of interleukin (IL)-4, IL-5, IL-10, and transforming growth factor-beta, whereas MS feeding counteracted this effect but enhanced IL-2, IL12p70, IL21, TNF-alpha, and interferon-gamma. In the SW480 colon adenocarcinoma cell-line, the cytotoxicity of spleenic T-cells from MS-fed animals was significantly increased. In the DMH-exposed rats, the expression of perforin in the spleenic T-cells was dramatically decreased, whereas MS abolished this depression. In summary, dietary MS suppresses DMH-induced immuno-imbalance as well as colon carcinogenesis in rats.

The main objective of this study was to investigate the activity of polydatin on mitochondrial dysfunction and lysosomal stability of arteriolar smooth muscle cells (ASMCs) in severe shock. The experimental animals (rats) were divided into five groups: control, hemorrhagic shock, shock + CsA, shock + Res, and shock + PD (exposed to cyclosporin A, resveratrol, or polydatin following induction of hemorrhagic shock, respectively). The calcein-Co(2+) technique revealed opening of ASMC mitochondrial permeability transition pores (mPTP) after shock with resulting mitochondrial swelling, decreased mitochondrial membrane potential (ΔΨm), and reduced intracellular ATP levels. These alterations were all inhibited by exposure to PD, which was significantly more effective than CsA and Res. PD also preserved lysosomal stability, suppressed activation of K(ATP) channels, ASMC hyperpolarization, and reduced vasoresponsiveness to norepinephrine that normally follows severe shock. The results demonstrate that exposure to PD after initiation of severe shock effectively preserves ASMC mitochondrial integrity and has a significant therapeutic effect in severe shock. The effects may partially result from lysosomal stabilization against shock-induced oxidative stress and depressed relocation of hydrolytic enzymes and redox-active lysosomal iron that, in turn, may induce mPTP opening.

Vitamin-B(12) is a generic term for corrinoid compounds that exhibit the biological activity of cyanocobalamin and are collectively referred to as cobalamins. Methylcobalamin and 5-deoxyadenosylcobalamin are the active cobalamins in human metabolism. Cobalamin plays a crucial role in the maintenance of homocysteine and methylmalonyl-CoA homeostasis and is required for erythrocyte formation and DNA synthesis. Data from human and animal studies indicate that cobalamin deficiency impairs neuronal function; a process that is thought to contribute to age-related cognitive decline and dementia. Cobalamin deficiency also results in dysfunction of the peripheral nervous system; among other disorders. Although there is a detailed understanding of the biochemical pathways that are perturbed in cobalamin deficiency, the mechanisms underlying age-related dyshomeostasis in such pathways remain to be addressed. Because cobalamin utilization is dependent on its efficient transit through lysosomes, and mounting evidence indicates that lysosomal function deteriorates in aging long-lived post-mitotic cells such as neurons, in the present article we review published data that supports the proposition that impaired lysosomal processing of cobalamin may play a significant role in age-related (neuro) degenerative diseases.

Iron is the most abundant transition metal in the earth's crust. It cycles easily between ferric (oxidized; Fe(III)) and ferrous (reduced; Fe(II)) and readily forms complexes with oxygen, making this metal a central player in respiration and related redox processes. However,'loose' iron, not within heme or iron-sulfur cluster proteins, can be destructively redox-active, causing damage to almost all cellular components, killing both cells and organisms. This may explain why iron is so carefully handled by aerobic organisms. Iron uptake from the environment is carefully limited and carried out by specialized iron transport mechanisms. One reason that iron uptake is tightly controlled is that most organisms and cells cannot efficiently excrete excess iron. When even small amounts of intracellular free iron occur, most of it is safely stored in a non-redox-active form in ferritins. Within nucleated cells, iron is constantly being recycled from aged iron-rich organelles such as mitochondria and used for construction of new organelles. Much of this recycling occurs within the lysosome, an acidic digestive organelle. Because of this, most lysosomes contain relatively large amounts of redox-active iron and are therefore unusually susceptible to oxidant-mediated destabilization or rupture. In many cell types, iron transit through the lysosomal compartment can be remarkably brisk. However, conditions adversely affecting lysosomal iron handling (or oxidant stress) can contribute to a variety of acute and chronic diseases. These considerations make normal and abnormal lysosomal handling of iron central to the understanding and, perhaps, therapy of a wide range of diseases.

The metal-chelating compound Dp44mT is a di-2-pyridylketone thiosemicarbazone (DpT) which displays potent and selective antitumor activity. This compound is receiving translational attention, but its mechanism is poorly understood. Here, we report that Dp44mT targets lysosome integrity through copper binding. Studies using the lysosomotropic fluorochrome acridine orange established that the copper-Dp44mT complex (Cu[Dp44mT]) disrupted lysosomes. This targeting was confirmed with pepstatin A-BODIPY FL, which showed redistribution of cathepsin D to the cytosol with ensuing cleavage of the proapoptotic BH3 protein Bid. Redox activity of Cu[Dp44mT] caused cellular depletion of glutathione, and lysosomal damage was prevented by cotreatment with the glutathione precursor N-acetylcysteine. Copper binding was essential for the potent antitumor activity of Dp44mT, as coincubation with nontoxic copper chelators markedly attenuated its cytotoxicity. Taken together, our studies show how the lysosomal apoptotic pathway can be selectively activated in cancer cells by sequestration of redox-active copper. Our findings define a novel generalized strategy to selectively target lysosome function for chemotherapeutic intervention against cancer.

Mustard seeds (MS), which are consumed in considerable amounts by the Japanese people that, interestingly, have the longest life expectancy in the world, are known to contain a number of yet not fully defined but quite powerful anti-oxidants. A suspension of extracted MS was found to suppress oxidized-LDL-induced macrophage respiratory burst in vitro, to prevent growth, and to induce apoptotic death of SW480 cells (a human colon cancer cell line), while no such effects were found for normal 3T3 cells. A diet enriched with MS decreased plasma levels of the lipid peroxidation product malonaldehyde in mice exposed to the colon cancer-inducer azoxymethane (AOM). Such a diet also dose-dependently enhanced the activity of several anti-oxidant enzymes, such as superoxide dismutase (SOD), catalase, and GSH-peroxidase and, moreover, reduced AOM-mediated formation of colon adenomas by about 50%. Further studies are required to detail and explore the beneficial effects of MS and their rich content of powerful anti-oxidants.

Motexafin gadolinium (MGd) sensitizes malignant cells to ionizing radiation, although the underlying mechanisms for uptake and sensitization are both unclear. Here we show that MGd is endocytosed by the clathrin-dependent pathway with ensuing lysosomal membrane permeabilization, most likely via formation of reactive oxygen species involving redox-active metabolites, such as ascorbate. We propose that subsequent apoptosis is a synergistic effect of irradiation and high MGd concentrations in malignant cells due to their pronounced endocytic activity. The results provide novel insights into the mode of action of this promising anti-cancer drug, which is currently under clinical trials.

To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.

Dihydrodichlorofluorescein (H2DCF-DA) is widely used to evaluate "cellular oxidative stress". After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol (H2DCF) that may be oxidized to fluorescent DCF by a process usually considered to involve reactive oxygen species (ROS). It is, however, not always recognized that, being a hydrophilic molecule, H2DCF does not pass membranes, except for the outer, fenestrated mitochondrial ones. It is also not generally realized that oxidation of H2DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor hydrogen peroxide, directly oxidizes H2DCF. Consequently, oxidation of H2DCF requires the presence of either cytochrome c or of both redox-active transition metals and hydrogen peroxide. Redox-active metals exist mainly within lysosomes, while cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H2DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to hydrogen peroxide, showing it by itself being unable to oxidize H2DCF. Cells that were either exposed to the lysosomotropic detergent MSDH, exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.