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Mitochondrial dysfunction and mutations in mitochondrial DNA have been implicated in a wide variety of human diseases, including cancer. In recent years, considerable advances in genomic, proteomic and bioinformatic technologies have made it possible the analysis of mitochondrial proteome, leading to the identification of over 1,000 proteins which have been assigned unambiguously to mitochondria. Defining the mitochondrial proteome is a fundamental step for fully understanding the organelle functions as well as mechanisms underlying mitochondrial pathology. In fact, besides giving information on mitochondrial physiology, by characterizing all the components of this subcellular organelle, the application of proteomic technologies permitted now to study the proteins involved in many crucial properties in cell signaling, cell differentiation and cell death and, in particular, to identify mitochondrial proteins that are aberrantly expressed in cancer cells. An improved understanding of the mitochondrial proteome could be essential to shed light on the connection between mitochondrial dysfunction, deregulation of apoptosis and tumorigenesis and to discovery new therapeutic targets for mitochondria-related diseases.

Anthracyclines remain the cornerstone in the treatment of many malignancies including lymphomas, leukaemias, and sarcomas. Unfortunately, the clinical use of these potent chemotherapeutics is severely limited by the development of a progressive dose-dependent cardiomyopathy that irreversibly evolves toward congestive heart failure. The molecular mechanisms responsible for anthracycline anticancer activity as well as those underlying anthracycline-induced cardiotoxicity are incompletely understood and remain a matter of remarkable controversy. Anthracyclines have long been considered to induce cardiotoxicity by mechanisms different from those mediating their anticancer activity. In particular, anthracycline antitumor efficacy is associated with nuclear DNA intercalation, topoisomerase II inhibition and drug-DNA adducts formation, whereas the cardiotoxicity is prevalently ascribed to oxidative stress and mitochondrial dysfunction. At present, however, the view that distinct mechanisms are implied in anticancer and cardiotoxic responses to anthracycline therapy does not seem fully convincing since beneficial (anticancer) and detrimental (cardiotoxic) effects are to some extent overlapping, share the subcellular organelle targets, the molecular effectors and the pathophysiological processes (i.e. DNA strand breaks, oxidative stress, signalling pathways, mitochondrial dysfunctions, apoptosis etc.).Here, we review the potential role of mitochondria in the molecular mechanisms underlying anthracyclines anticancer activity as well as in the pathogenesis of anthracycline-induced cardiotoxicity.

The evolving role of mitochondria as a target for different death-inducing noxae prompted us to investigate trimethyltin (TMT)-dependent effects on mitochondrial functionality. For this purpose, we used a homogeneous cell culture model represented by undifferentiated PC12 cells. Mitochondria isolated from PC12 cells treated with TMT for 6, 12 and 24h, showed a time-dependent inhibition of ADP-stimulated oxygen consumption using succinate or glutamate/malate as substrate. Using a fluorescent assay, the effect of TMT on mitochondrial membrane potential (delta Psi) in PC12 cells was also determined. After 24h in culture, a strong loss of mitochondrial membrane potential (delta Psi) was observed in TMT-treated cells. Collapse of mitochondrial membrane potential correlated with an increased expression of bax/bcl-2 ratio, as evaluated by polymerase chain reaction. Western blotting and spectrophotometric analysis showed that cytochrome c release and activation of caspase 3 were concurrently induced. Our findings suggest that inhibition of mitochondrial respiration represents the early toxic event for cell death in PC12 due to trimethyltin.

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear receptors now actively investigated for their involvement in lipid and glucidic metabolism, immune regulation and cell differentiation. Drugs binding and activating PPARs are therefore attracting attention for their potential therapeutic role in various diseases like type 2 diabetes, dyslipidemias, atherosclerosis, obesity (i.e., metabolic syndrome). Agonists of these receptors have been already used in therapeutic protocols: fibrates (PPAR-alpha ligands) are being used in hyperlipidemias, and thiazolidinediones (mainly PPAR-gamma ligands) are being employed as insulin sensitizers. The latter drugs introduction into therapy, however, showed very soon some unwanted effects (hepatotoxicity at first and myocardiotoxicity later on) which confirmed some contradictory data already suggested by pre-clinical trial-experiments. In this study we show that some PPAR ligands impair mitochondrial oxidative metabolism in human liver cell line mainly by deranging NADH oxidation. Intriguingly, the PPAR-gamma ligand ciglitazone caused a dose-dependent inhibition of NADH-cytochrome c reductase that resulted, at a drug concentration of 50 microM, of about 60% (P<0.001), while other PPAR ligands with different receptor affinity - positive controls like clofibrate (0.7 mM), gemfibrozil (0.23 mM) and bezafibrate (1 mM) - reduced the activity of mitochondrial Complex I by about 20% (P<0.01, P<0.01 and P<0.05, respectively). The induced mitochondrial dysfunction imposed a series of metabolic compensatory adaptations characterized by a significant shift to anaerobic glycolysis. These findings underline the undervalued non-genomic effects of PPAR ligands and can provide a better understanding of the pharmacotoxicological profiles of these drugs and of their roles in the therapy of diabetes mellitus.

In addition to their well-known critical role in energy metabolism, mitochondria are now recognized as the location where various catabolic and anabolic processes, calcium fluxes, various oxygen-nitrogen reactive species, and other signal transduction pathways interact to maintain cell homeostasis and to mediate cellular responses to different stimuli. It is important to consider how pharmacological agents affect mitochondrial biochemistry, not only because of toxicological concerns but also because of potential therapeutic applications. Several potential targets could be envisaged at the mitochondrial level that may underlie the toxic effects of some drugs. Recently, antiviral nucleoside analogs have displayed mitochondrial toxicity through the inhibition of DNA polymerase-gamma (pol-gamma). Other drugs that target different components of mitochondrial channels can disrupt ion homeostasis or interfere with the mitochondrial permeability transition pore. Many known inhibitors of the mitochondrial electron transfer chain act by interfering with one or more of the respiratory chain complexes. Nonsteroidal anti-inflammatory drugs (NSAIDs), for example, may behave as oxidative phosphorylation uncouplers. The mitochondrial toxicity of other drugs seems to depend on free radical production, although the mechanisms have not yet been clarified. Meanwhile, drugs targeting mitochondria have been used to treat mitochondrial dysfunctions. Importantly, drugs that target the mitochondria of cancer cells have been developed recently; such drugs can trigger apoptosis or necrosis of the cancer cells. Thus the aim of this review is to highlight the role of mitochondria in pharmacotoxicology, and to describe whenever possible the main molecular mechanisms underlying unwanted and/or therapeutic effects.

PPARs are a class of nuclear receptors involved in lipid and glucidic metabolism, immune regulation and cell differentiation. This spectrum of biological activities stimulated pharmacological research to synthetize different molecules with PPARs binding activity with beneficial therapeutic effects. As a matter of fact, some synthetic PPAR-ligands have been already employed in pharmacotherapy: PPAR-alpha ligands, such as fibrates, are used in hyperlipidemias and thiazolidinediones, mainly PPAR-gamma ligands, are employed as insulin sensitizers. However, both classes of drugs showed pharmacotoxicological profiles which cannot be fully ascribed to activation of their specific receptors and which are causing a growing incidence of dramatic side effects (rhabdomyolysis, acute liver failure, heart failure, etc.). A re-evaluation of the biological activities of PPAR synthetic ligands, in particular of the mitochondrial dysfunction based on a rotenone-like Complex I partial inhibition and of its consequent metabolic adaptations, seems to explain some of the pathophysiologic aspects of PPARs allowing a better definition of the therapeutic properties of the so-called PPAR-ligands.

Experimental hyperoxia represents a suitable in vitro model to study some pathogenic mechanisms related to oxidative stress. Moreover, it allows the investigation of the molecular pathophysiology underlying oxygen therapy and toxicity. In this study, a modified experimental set up was adopted to accomplish a model of moderate hyperoxia (50% O(2), 96 h culture) to induce oxidative stress in the human leukemia cell line, U-937. Spectrophotometric measurements of mitochondrial respiratory enzyme activities, NMR spectroscopy of culture media, determination of antioxidant enzyme activities, and cell proliferation and differentiation assays were performed. The data showed that moderate hyperoxia in this myeloid cell line causes: i) intriguing alterations in the mitochondrial activities at the levels of succinate dehydrogenase and succinate-cytochrome c reductase; ii) induction of metabolic compensatory adaptations, with significant shift to glycolysis; iii) induction of different antioxidant enzyme activities; iv) significant cell growth inhibition and v) no significant apoptosis. This work will permit better characterization the mitochondrial damage induced by hyperoxia. In particular, the data showed a large increase in the succinate cytochrome c reductase activity, which could be a fundamental pathogenic mechanism at the basis of oxygen toxicity.

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear receptors involved in lipid and glucidic metabolism, immune regulation, and cell differentiation. Many of their biological activities have been studied by using selective synthetic activators (mainly fibrates and thiazolidinediones) which have been already employed in therapeutic protocols. Both kinds of drugs, however, showed pharmacotoxicological profiles, which cannot be ascribed by any means to receptor activation. To better understand these non-receptorial or extrareceptorial aspects, the effect of different PPAR-ligands on the metabolic status of human HL-60 cell line has been investigated. At this regard, NMR analysis of cell culture supernatants was accomplished in order to monitor modifications at the level of cell metabolism. Cell growth and chemiluminescence assays were employed to verify cell differentiation. Results showed that all the considered PPAR-ligands, although with different potencies and independently from their PPAR binding specificity, induced a significant derangement of the mitochondrial respiratory chain consisting in a strong inhibition of NADH-cytochrome c reductase activity. This derangement has been shown to be strictly correlated to the adaptive metabolic modifications, as evidenced by the increased formation of lactate and acetate, due to the stimulation of anaerobic glycolysis and fatty acid beta-oxidation. It is worthy noting that the mitochondrial dysfunction appeared also linked to the capacity of any given PPAR-ligand to induce cell differentiation. These data could afford an explanation of biochemical and toxicological aspects related to the therapeutic use of synthetic PPAR-ligands and suggest a revision of PPAR pathophysiologic mechanisms.

Abstract: Drug induced cell differentiation represents a promising experimental model for proteomic analysis of cancer cells. In fact, by modulating and monitoring neoplastic cell differentiation it could be possible to identify cytodifferentiation related protein expression changes that can be subsequently utilized in vivo as potential cancer biomarkers. One main advantage of this approach is the significant reduction of biological variability normally observed in clinical biomarker research, with important implications also in prognosis and therapy. At this regard, a new class of differentiating agents is emerging, the so called PPAR-ligands, which however are characterized by a debated mechanism of action that has not been yet studied through a proteomic approach. To this aim, we investigated ciglitazone-induced differentiation of a human hepatocarcinoma HepG2 cell line, by monitoring biochemical and cellular parameters of cytodifferentiation and modifications of cellular protein profiles through 2-DE and MALDI-TOF analysis. Independent of the hypothesized mechanism of action of this intriguing PPARγ agonist, results indicated that ciglitazone is a strong differentiating agent for the HepG2 cell line and that this process is associated with modifications of protein expression related to cell antioxidant systems, the cell cycle apparatus, signal transduction pathways, cellular stress and invasiveness. At last, considering these and other published data, a proteomic profile related to the cancer aggressiveness is beginning to emerge. [Copyright &y& Elsevier]

Abstract: In order to clarify the basis of neuronal toxicity exerted by the shortest active peptides of amyloid beta-protein (Aβ), the toxic effects of Aβ(31–35) and Aβ(25–35) peptides on isolated rat brain mitochondria were investigated. The results show that exposure of isolated rat brain mitochondria to Aβ(31–35) and Aβ(25–35) peptides determines: (i) release of cytochrome c; (ii) mitochondrial swelling and (iii) a significant reduction in mitochondrial oxygen consumption. In contrast, the amplitude of these events resulted attenuated in isolated brain mitochondria exposed to the Aβ(31–35)Met35OX in which methionine-35 was oxidized to methionine sulfoxide. The Aβ peptide derivative with norleucine substituting Met-35, i.e., Aβ(31–35)Nle-35, had not effect on any of the biochemical parameters tested. We have further characterized the action of Aβ(31–35) and Aβ(25–35) peptides on neuronal cells. Taken together our result indicate that Aβ(31–35) and Aβ(25–35) peptides in non-aggregated form, i.e., predominantly monomeric, are strongly neurotoxic, having the ability to enter within the cells, determining mitochondrial damage with an evident trigger of apoptotic signals. Such a mechanism of toxicity seems to be dependent by the redox state of methionine-35. [Copyright &y& Elsevier]

Abstract: The toxic behaviour of the two shorter sequences of the native Aβ amyloid peptide required for cytotoxicity i.e., Aβ(31–35) and Aβ(25–35) peptides, was studied. We have shown that Aβ(31–35) peptide induces neurotoxicity in undifferentiated PC 12 cell via an apoptotic cell death pathway, including caspase activation and DNA fragmentation. Aβ(25–35) peptide, like the shorter amyloid peptide has the ability to induce neurotoxicity, as evaluated by the MTS reduction assay and by adherent cell count, but the Aβ(25–35) peptide-induced neurotoxicity is not associated with any biochemical features of apoptosis. The differences observed between the neurotoxic properties of Aβ(31–35) and Aβ(25–35) peptides might result on their different ability to be internalised within the neuronal cells. Furthermore, this study reveals that the redox state of methionine residue, C-terminal in Aβ(31–35) and Aβ(25–35) peptides affect in a different way the toxic behaviour of these two short amyloid fragments. Taken together our results suggest that Aβ(31–35) peptide induces cell death by apoptosis, unlike the Aβ(25–35) peptide and that role played by methionine-35 in Aβ induced neurotoxicity might be related to the Aβ aggregation state. [Copyright &y& Elsevier]