Early effects of the antineoplastic agent salinomycin on mitochondrial function.

Salinomycin, isolated from Streptomyces albus, displays antimicrobial activity. Recently, a large-scale screening approach identified salinomycin and nigericin as selective apoptosis inducers of cancer stem cells. Growing evidence suggests that salinomycin is able to kill different types of non-stem tumor cells that usually display resistance to common therapeutic approaches, but the mechanism of action of this molecule is still poorly understood. Since salinomycin has been suggested to act as a K(+) ionophore, we explored its impact on mitochondrial bioenergetic performance at an early time point following drug application. In contrast to the K(+) ionophore valinomycin, salinomycin induced a rapid hyperpolarization. In addition, mitochondrial matrix acidification and a significant decrease of respiration were observed in intact mouse embryonic fibroblasts (MEFs) and in cancer stem cell-like HMLE cells within tens of minutes, while increased production of reactive oxygen species was not detected. By comparing the chemical structures and cellular effects of this drug with those of valinomycin (K(+) ionophore) and nigericin (K(+)/H(+) exchanger), we conclude that salinomycin mediates K(+)/H(+) exchange across the inner mitochondrial membrane. Compatible with its direct modulation of mitochondrial function, salinomycin was able to induce cell death also in Bax/Bak-less double-knockout MEF cells. Since at the concentration range used in most studies (around 10 µM) salinomycin exerts its effect at the level of mitochondria and alters bioenergetic performance, the specificity of its action on pathologic B cells isolated from patients with chronic lymphocytic leukemia (CLL) versus B cells from healthy subjects was investigated. Mesenchymal stromal cells (MSCs), proposed to mimic the tumor environment, attenuated the apoptotic effect of salinomycin on B-CLL cells. Apoptosis occurred to a significant extent in healthy B cells as well as in MSCs and human primary fibroblasts. The results indicate that salinomycin, when used above µM concentrations, exerts direct, mitochondrial effects, thus compromising cell survival.

Design and synthesis of new bifunctional sigma-1 selective ligands with antioxidant activity.

Herein we report the synthesis of new bifunctional sigma-1 (σ1)-selective ligands with antioxidant activity. To achieve this goal, we combined the structure of lipoic acid, a universal antioxidant, with an appropriate sigma aminic moiety. Ligands 14 and 26 displayed high affinity and selectivity for σ1 receptors (Kiσ1 = 1.8 and 5.5 nM; Kiσ2/σ1 = 354 and 414, respectively). Compound 26 exhibited in vivo antiopioid effects on kappa opioid (KOP) receptor-mediated analgesia. In rat liver and brain mitochondria (RLM, RBM), this compound significantly reduced the swelling and the oxidation of thiol groups induced by calcium ions. Our results demonstrate that the tested compound has protective effects against oxidative stress.

Interactions of melatonin with mammalian mitochondria. Reducer of energy capacity and amplifier of permeability transition.

Melatonin, a metabolic product of the amino acid tryptophan, induces a dose-dependent energy drop correlated with a decrease in the oxidative phosphorylation process in isolated rat liver mitochondria. This effect involves a gradual decrease in the respiratory control index and significant alterations in the state 4/state 3 transition of membrane potential (ΔΨ). Melatonin, alone, does not affect the insulating properties of the inner membrane but, in the presence of supraphysiological Ca2+, induces a ΔΨ drop and colloid-osmotic mitochondrial swelling. These events are sensitive to cyclosporin A and the inhibitors of Ca2+ transport, indicative of the induction or amplification of the mitochondrial permeability transition. This phenomenon is triggered by oxidative stress induced by melatonin and Ca2+, with the generation of hydrogen peroxide and the consequent oxidation of sulfydryl groups, glutathione and pyridine nucleotides. In addition, melatonin, again in the presence of Ca2+, can also induce substantial release of cytochrome C and AIF (apoptosis-inducing factor), thus revealing its potential as a pro-apoptotic agent.

Potential anticancer application of polyamine oxidation products formed by amine oxidase: a new therapeutic approach.

The polyamines spermine, spermidine and putrescine are ubiquitous cell components. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper-containing amine oxidases. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. In tumors, polyamines and amine oxidases are increased as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H(2)O(2) and aldehydes produced by the reaction. This study demonstrated that multidrug-resistant (MDR) cancer cells (colon adenocarcinoma and melanoma) are significantly more sensitive than the corresponding wild-type (WT) ones to H(2)O(2) and aldehydes, the products of BSAO-catalyzed oxidation of spermine. Transmission electron microscopy (TEM) observations showed major ultrastructural alterations of the mitochondria. These were more pronounced in MDR than in WT cells. Increasing the incubation temperature from 37 to 42 degrees Celsius enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumor growth, particularly well if the enzyme has been conjugated to a biocompatible hydrogel polymers. Since both wild-type and MDR cancer cells after pre-treatment with MDL 72527, a lysosomotropic compound, are sensitized to subsequent exposure to BSAO/spermine, it is conceivable that combined treatment with a lysosomotropic compound and BSAO/spermine would be effective against tumor cells. It is of interest to search for such novel compounds, which might be promising for application in a therapeutic setting.

Agmatine prevents the Ca(2+)-dependent induction of permeability transition in rat brain mitochondria.

The arginine metabolite agmatine is able to protect brain mitochondria against the drop in energy capacity by the Ca(2+)-dependent induction of permeability transition (MPT) in rat brain mitochondria. At normal levels, the amine maintains the respiratory control index and ADP/O ratio and prevents mitochondrial colloid-osmotic swelling and any electrical potential (DeltaPsi) drop. MPT is due to oxidative stress induced by the interaction of Ca(2+) with the mitochondrial membrane, leading to the production of hydrogen peroxide and, subsequently, other reactive oxygen species (ROS) such as hydroxyl radicals. This production of ROS induces oxidation of sulfhydryl groups, in particular those of two critical cysteines, most probably located on adenine nucleotide translocase, and also oxidation of pyridine nucleotides, resulting in transition pore opening. The protective effect of agmatine is attributable to a scavenging effect on the most toxic ROS, i.e., the hydroxyl radical, thus preventing oxidative stress and consequent bioenergetic collapse.

Polyamines: fundamental characters in chemistry and biology.

Polyamines are small cationic molecules required for cellular proliferation and are detected at higher concentrations in most tumour tissues, compared to normal tissues. Agmatine (AGM), a biogenic amine, is able to arrest proliferation in cell lines by depleting intracellular polyamine levels. It enters mammalian cells via the polyamine transport system. Agmatine is able to induce oxidative stress in mitochondria at low concentrations (10 or 100 microM), while at higher concentrations (e.g. 1-2 mM) it does not affect mitochondrial respiration and is ineffective in inducing any oxidative stress. As this effect is strictly correlated with the mitochondrial permeability transition induction and the triggering of the pro-apoptotic pathway, AGM may be considered as a regulator of this type of cell death. Furthermore, polyamine transport is positively correlated with the rate of cellular proliferation. By increasing the expression of antizyme, a protein that inhibits polyamine biosynthesis and transport, AGM also exhibits a regulatory effect on cell proliferation. Methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosyl-L: -methionine decarboxylase, displaying anticancer activity, is a structural analogue of the natural polyamine spermidine. MGBG has been extensively studied, preclinically as well as clinically, and its anticancer activity has been attributed to the inhibition of polyamine biosynthesis and also to its effect on mitochondrial function. Numerous findings have suggested that MGBG might be used as a chemotherapeutic agent against cancer.

Agmatine transport in brain mitochondria: a different mechanism from that in liver mitochondria.

The diamine agmatine (AGM), exhibiting two positive charges at physiological pH, is transported into rat brain mitochondria (RBM) by an electrophoretic mechanism, requiring high membrane potential values and exhibiting a marked non-ohmic force-flux relationship. The mechanism of this transport apparently resembles that observed in rat liver mitochondria (RLM), but there are several characteristics that strongly suggest the presence of a different transporter of agmatine in RBM. In this type of mitochondria, the extent of initial binding and total accumulation is higher and lower, respectively, than that in liver; saturation kinetics and the flux-voltage relationship also exhibit different trends, whereas idazoxan and putrescine, ineffective in RLM, act as inhibitors. The characteristics of agmatine uptake in RBM lead to the conclusion that its transporter is a channel with two asymmetric energy barriers, showing some characteristics similar to those of the imidazoline receptor I(2) and the sharing with the polyamine transporter.