Incorporating a Polyethyleneglycol Linker to Enhance the Hydrophilicity of Mitochondria-Targeted Triphenylphosphonium Constructs

The targeting of bioactive molecules and probes to mitochondria can be achieved by coupling to the lipophilic triphenyl phosphonium (TPP) cation, which accumulates several hundred-fold within mitochondria in response to the mitochondrial membrane potential (Δψ_m). Typically, a simple alkane links the TPP to its “cargo”, increasing overall hydrophobicity. As it would be beneficial to enhance the water solubility of mitochondria-targeted compounds we explored the effects of replacing the alkyl linker with a polyethylene glycol (PEG). We found that the use of PEG led to compounds that were readily taken up by isolated mitochondria and by mitochondria inside cells. Within mitochondria the PEG linker greatly decreased adsorption of the TPP constructs to the matrix-facing face of the mitochondrial inner membrane. These findings will allow the distribution of mitochondria-targeted TPP compounds within mitochondria to be fine-tuned.     

Mitochondrial Membrane Disrupting Molecules for Selective Killing of Senescent Cells

Cellular senescence, a stable form of cell cycle arrest, facilitates protection from tumorigenesis and aids in tissue repair as they accumulate in the body at an early age. However, long-term retention of senescent cells causes inflammation, aging of the tissue, and progression of deadly diseases such as obesity, diabetes, and atherosclerosis. Various attempts have been made to achieve selective elimination of senescent cells from the body, yet little has been explored in designing the mitochondria-targeted senolytic agent. Many characteristics of senescence are associated with mitochondria. Here we have designed a library of alkyl-monoquaternary ammonium-triphenyl phosphine (TPP) and alkyl-diquaternary ammonium-TPP of varying alkyl chain lengths, which target the mitochondria; we also studied their senolytic properties. It was observed that the alkyl-diquaternary ammonium-TPP with the longest chain length induced apoptosis in senescent cells selectively via an increase of reactive oxygen species (ROS) and mitochondrial membrane disruption. This study demonstrates that mitochondria could be a potential target for designing new small molecules as senolytic agents for the treatment of a variety of dysfunctions associated with pathological aging.     

Mitochondria-Derived Reactive Oxygen Species Play an Important Role in Doxorubicin-Induced Platelet Apoptosis

Doxorubicin (DOX) is an effective chemotherapeutic agent; however; its use is limited by some side effects; such as cardiotoxicity and thrombocytopenia. DOX-induced cardiotoxicity has been intensively investigated; however; DOX-induced thrombocytopenia has not been clearly elucidated. Here we show that DOX-induced mitochondria-mediated intrinsic apoptosis and glycoprotein (GP)Ibα shedding in platelets. DOX did not induce platelet activation; whereas; DOX obviously reduced adenosine diphosphate (ADP)- and thrombin-induced platelet aggregation; and impaired platelet adhesion on the von Willebrand factor (vWF) surface. In addition; we also show that DOX induced intracellular reactive oxygen species (ROS) production and mitochondrial ROS generation in a dose-dependent manner. The mitochondria-targeted ROS scavenger Mito-TEMPO blocked intracellular ROS and mitochondrial ROS generation. Furthermore; Mito-TEMPO reduced DOX-induced platelet apoptosis and GPIbα shedding. These data indicate that DOX induces platelet apoptosis; and impairs platelet function. Mitochondrial ROS play a pivotal role in DOX-induced platelet apoptosis and GPIbα shedding. Therefore; DOX-induced platelet apoptosis might contribute to DOX-triggered thrombocytopenia; and mitochondria-targeted ROS scavenger would have potential clinical utility in platelet-associated disorders involving mitochondrial oxidative damage.     

Mitochondrion as a Target of Astaxanthin Therapy in Heart Failure

Mitochondria are considered to be important organelles in the cell and play a key role in the physiological function of the heart, as well as in the pathogenesis and development of various heart diseases. Under certain pathological conditions, such as cardiovascular diseases, stroke, traumatic brain injury, neurodegenerative diseases, muscular dystrophy, etc., mitochondrial permeability transition pore (mPTP) is formed and opened, which can lead to dysfunction of mitochondria and subsequently to cell death. This review summarizes the results of studies carried out by our group of the effect of astaxanthin (AST) on the functional state of rat heart mitochondria upon direct addition of AST to isolated mitochondria and upon chronic administration of AST under conditions of mPTP opening. It was shown that AST exerted a protective effect under all conditions. In addition, AST treatment was found to prevent isoproterenol-induced oxidative damage to mitochondria and increase mitochondrial efficiency. AST, a ketocarotenoid, may be a potential mitochondrial target in therapy for pathological conditions associated with oxidative damage and mitochondrial dysfunction, and may be a potential mitochondrial target in therapy for pathological conditions.     

Targeting Lysosomes to Reverse Hydroquinone-Induced Autophagy Defects and Oxidative Damage in Human Retinal Pigment Epithelial Cells

In age-related macular degeneration (AMD), hydroquinone (HQ)-induced oxidative damage in retinal pigment epithelium (RPE) is believed to be an early event contributing to dysregulation of inflammatory cytokines and vascular endothelial growth factor (VEGF) homeostasis. However, the roles of antioxidant mechanisms, such as autophagy and the ubiquitin-proteasome system, in modulating HQ-induced oxidative damage in RPE is not well-understood. This study utilized an in-vitro AMD model involving the incubation of human RPE cells (ARPE-19) with HQ. In comparison to hydrogen peroxide (H₂O₂), HQ induced fewer reactive oxygen species (ROS) but more oxidative damage as characterized by protein carbonyl levels, mitochondrial dysfunction, and the loss of cell viability. HQ blocked the autophagy flux and increased proteasome activity, whereas H₂O₂ did the opposite. Moreover, the lysosomal membrane-stabilizing protein LAMP2 and cathepsin D levels declined with HQ exposure, suggesting loss of lysosomal membrane integrity and function. Accordingly, HQ induced lysosomal alkalization, thereby compromising the acidic pH needed for optimal lysosomal degradation. Pretreatment with MG132, a proteasome inhibitor and lysosomal stabilizer, upregulated LAMP2 and autophagy and prevented HQ-induced oxidative damage in wildtype RPE cells but not cells transfected with shRNA against ATG5. This study demonstrated that lysosomal dysfunction underlies autophagy defects and oxidative damage induced by HQ in human RPE cells and supports lysosomal stabilization with the proteasome inhibitor MG132 as a potential remedy for oxidative damage in RPE and AMD.     

Biomarkers and Mechanisms of Oxidative Stress—Last 20 Years of Research with an Emphasis on Kidney Damage and Renal Transplantation

Oxidative stress is an imbalance between pro- and antioxidants that adversely influences the organism in various mechanisms and on many levels. Oxidative damage occurring concomitantly in many cellular structures may cause a deterioration of function, including apoptosis and necrosis. The damage leaves a molecular “footprint”, which can be detected by specific methodology, using certain oxidative stress biomarkers. There is an intimate relationship between oxidative stress, inflammation, and functional impairment, resulting in various diseases affecting the entire human body. In the current narrative review, we strengthen the connection between oxidative stress mechanisms and their active compounds, emphasizing kidney damage and renal transplantation. An analysis of reactive oxygen species (ROS), antioxidants, products of peroxidation, and finally signaling pathways gives a lot of promising data that potentially will modify cell responses on many levels, including gene expression. Oxidative damage, stress, and ROS are still intensively exploited research subjects. We discuss compounds mentioned earlier as biomarkers of oxidative stress and present their role documented during the last 20 years of research. The following keywords and MeSH terms were used in the search: oxidative stress, kidney, transplantation, ischemia-reperfusion injury, IRI, biomarkers, peroxidation, and treatment.     

Treatment Strategies that Enhance the Efficacy and Selectivity of Mitochondria-Targeted Anticancer Agents

Nearly a century has passed since Otto Warburg first observed high rates of aerobic glycolysis in a variety of tumor cell types and suggested that this phenomenon might be due to an impaired mitochondrial respiratory capacity in these cells. Subsequently, much has been written about the role of mitochondria in the initiation and/or progression of various forms of cancer, and the possibility of exploiting differences in mitochondrial structure and function between normal and malignant cells as targets for cancer chemotherapy. A number of mitochondria-targeted compounds have shown efficacy in selective cancer cell killing in pre-clinical and early clinical testing, including those that induce mitochondria permeability transition and apoptosis, metabolic inhibitors, and ROS regulators. To date, however, none has exhibited the standards for high selectivity and efficacy and low toxicity necessary to progress beyond phase III clinical trials and be used as a viable, single modality treatment option for human cancers. This review explores alternative treatment strategies that have been shown to enhance the efficacy and selectivity of mitochondria-targeted anticancer agents in vitro and in vivo, and may yet fulfill the clinical promise of exploiting the mitochondrion as a target for cancer chemotherapy.     

Modulation of Cellular Fate of Vinyl Triarylamines through Structural Fine Tuning: To Stay or Not To Stay in the Mitochondria?

Mitochondria are involved in many cellular pathways and dysfunctional mitochondria are linked to various diseases. Hence efforts have been made to design mitochondria-targeted fluorophores for monitoring the mitochondrial status. However, the factors that govern the mitochondria-targeted potential of dyes are not well-understood. In this context, we synthesized analogues of the TP-2Bzim probe belonging to the vinyltriphenylamine (TPA) class and already described for its capacity to bind nuclear DNA in fixed cells and mitochondria in live cells. These analogues ( TP-1Bzim, TPⁿ-2Bzim, TP¹⁺-2Bzim, TN-2Bzim ) differ in the cationic charge, the number of vinylbenzimidazolium branches and the nature of the triaryl core. Using microscopy, we demonstrated that the cationic derivatives accumulate in mitochondria but do not reach mtDNA. Under depolarisation of the mitochondrial membrane, TP-2Bzim and TP¹⁺-2Bzim translocate to the nucleus in direct correlation with their strong DNA affinity. This reversible phenomenon emphasizes that these probes can be used to monitor ΔΨ_m variations.     

New Biocompatible β-Phosphorylated Linear Nitrones Targeting Mitochondria: Protective Effect in Apoptotic Cells

The mitochondrion, an essential organelle involved in cellular respiration, energy production, and cell death, is the main cellular source of reactive oxygen species (ROS), including superoxide. Mitochondrial diseases resulting from uncontrolled/excess ROS generation are an emerging public health concern and there is current interest in specific mitochondriotropic probes to get information on in-situ ROS production. As such, nitrones vectorized by the triphenylphosphonium (TPP) cation have recently drawn attention despite reported cytotoxicity. Herein, we describe the synthesis of 13 low-toxic derivatives of N-benzylidene-1-diethoxyphosphoryl-1-methylethylamine N-oxide (PPN) alkyl chain-grafted to a pyridinium, triethylammonium or berberinium lipophilic cation. These nitrones showed in-vitro superoxide quenching activity and EPR/spin-trapping efficiency towards biologically relevant free radicals, including superoxide and hydroxyl radicals. Their mitochondrial penetration was confirmed by ³¹P NMR spectroscopy, and their anti-apoptotic properties were assessed in Schwann cells treated with hydrogen peroxide. Two pyridinium-substituted PPNs were identified as potentially better alternatives to TPP nitrones conjugates for studying mitochondrial oxidative damage.     

Effect of antioxidant contained in poly(ethylene glycol) on cell fusion

The effect of antioxidants contained in poly(ethylene glycol) (PEG) on cell fusion was studied using L929 cells in the monolayer state. Hydroquinone monomethyl ether (HQME), 2-mercaptobenzimidazole (MB), butyl hydroxyanisole (BHA) and 2,6-di-(t-butyl)-4-methylphenol (BHT) were chosen from the antioxidants that have currently been used to protect commercially available PEG from oxidation. Cell culture was conducted in 40% w/w aqueous solution of PEG with a molecular weight of 3000 in the presence of different concentrations of antioxidants. BHA clearly enhanced membrane fusion of L929 cells with increasing concentration in PEG solution, whereas HQME, MB and BHT had no significant effect on cell fusion. The enhancement of cell fusion by BHA might be ascribed to balanced hydrophobicity and high water solubility in comparison with the other three antioxidants.     

Oxidative Stress in the Pathogenesis of Keratoconus and Fuchs Endothelial Corneal Dystrophy

Due to its localization and function, the cornea is regularly exposed to sunlight and atmospheric oxygen, mainly dioxygen, which produce reactive oxygen species (ROS). Therefore, corneal cells are particularly susceptible to oxidative stress. The accumulation of ROS in the cornea may affect signal transduction, proliferation and may also promote cell death. The cornea has several enzymatic and non-enzymatic antioxidants involved in ROS scavenging, but in certain conditions they may not cope with oxidative stress, leading to diseases of the eye. Keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD) are multifactorial diseases of the cornea, in which pathogenesis is not fully understood. However, increased levels of oxidative stress markers detected in these disorders indicate that oxidative stress may play an important role in their development and progression. These markers are: (i) decreased levels of non-enzymatic antioxidants, and (ii) decreased expression of genes encoding antioxidative enzymes, including thioredoxin reductase, peroxiredoxins, superoxide dismutase, glutathione S-transferase, and aldehyde dehydrogenase. Moreover, the FECD endothelium displays higher levels of oxidative DNA damage, especially in mitochondrial DNA (mtDNA), whereas KC cornea shows abnormal levels of some components of oxidative phosphorylation encoded by mtDNA. In this review we present some considerations and results of experiments supporting the thesis on the important role of oxidative stress in KC and FECD pathology.     

MiR-25 Protects Cardiomyocytes against Oxidative Damage by Targeting the Mitochondrial Calcium Uniporter

MicroRNAs (miRNAs) are a class of small non-coding RNAs, whose expression levels vary in different cell types and tissues. Emerging evidence indicates that tissue-specific and -enriched miRNAs are closely associated with cellular development and stress responses in their tissues. MiR-25 has been documented to be abundant in cardiomyocytes, but its function in the heart remains unknown. Here, we report that miR-25 can protect cardiomyocytes against oxidative damage by down-regulating mitochondrial calcium uniporter (MCU). MiR-25 was markedly elevated in response to oxidative stimulation in cardiomyocytes. Further overexpression of miR-25 protected cardiomyocytes against oxidative damage by inactivating the mitochondrial apoptosis pathway. MCU was identified as a potential target of miR-25 by bioinformatical analysis. MCU mRNA level was reversely correlated with miR-25 under the exposure of H₂O₂, and MCU protein level was largely decreased by miR-25 overexpression. The luciferase reporter assay confirmed that miR-25 bound directly to the 3'' untranslated region (UTR) of MCU mRNA. MiR-25 significantly decreased H₂O₂-induced elevation of mitochondrial Ca²⁺ concentration, which is likely to be the result of decreased activity of MCU. We conclude that miR-25 targets MCU to protect cardiomyocytes against oxidative damages. This finding provides novel insights into the involvement of miRNAs in oxidative stress in cardiomyocytes.     

Endothelial Aging Associated with Oxidative Stress Can Be Modulated by a Healthy Mediterranean Diet

Aging is a condition which favors the development of atherosclerosis, which has been associated with a breakdown in repair processes that occurs in response to cell damage. The dysregulation of the biological systems associated with aging are produced partly through damage which accumulates over time. One major source of this injury is oxidative stress, which can impair biological structures and the mechanisms by which they are repaired. These mechanisms are based on the pathogenesis of endothelial dysfunction, which in turn is associated with cardiovascular disease, carcinogenesis and aging. The dependent dysfunction of aging has been correlated with a reduction in the number and/or functional activity of endothelial progenitor cells, which could hinder the repair and regeneration of the endothelium. In addition, aging, inflammation and oxidative stress are endogenous factors that cause telomere shortening, which is dependent on oxidative cell damage. Moreover, telomere length correlates with lifestyle and the consumption of a healthy diet. Thus, diseases associated with aging and age may be caused by the long-term effects of oxidative damage, which are modified by genetic and environmental factors. Considering that diet is a very important source of antioxidants, in this review we will analyze the relationship between oxidative stress, aging, and the mechanisms which may be involved in a higher survival rate and a lower incidence of the diseases associated with aging in populations which follow a healthy diet.     

Medicinal Chemistry Targeting Mitochondria: From New Vehicles and Pharmacophore Groups to Old Drugs with Mitochondrial Activity

Interest in tumor cell mitochondria as a pharmacological target has been rekindled in recent years. This attention is due in part to new publications documenting heterogenous characteristics of solid tumors, including anoxic and hypoxic zones that foster cellular populations with differentiating metabolic characteristics. These populations include tumor-initiating or cancer stem cells, which have a strong capacity to adapt to reduced oxygen availability, switching rapidly between glycolysis and oxidative phosphorylation as sources of energy and metabolites. Additionally, this cell subpopulation shows high chemo- and radioresistance and a high capacity for tumor repopulation. Interestingly, it has been shown that inhibiting mitochondrial function in tumor cells affects glycolysis pathways, cell bioenergy, and cell viability. Therefore, mitochondrial inhibition may be a viable strategy for eradicating cancer stem cells. In this context, medicinal chemistry research over the last decade has synthesized and characterized “vehicles” capable of transporting novel or existing pharmacophores to mitochondrial tumor cells, based on mechanisms that exploit the physicochemical properties of the vehicles and the inherent properties of the mitochondria. The pharmacophores, some of which have been isolated from plants and others, which were synthesized in the lab, are diverse in chemical nature. Some of these molecules are active, while others are prodrugs that have been evaluated alone or linked to mitochondria-targeted agents. Finally, researchers have recently described drugs with well-proven safety and efficacy that may exert a mitochondria-specific inhibitory effect in tumor cells through noncanonical mechanisms. The effectiveness of these molecules may be improved by linking them to mitochondrial carrier molecules. These promising pharmacological agents should be evaluated alone and in combination with classic chemotherapeutic drugs in clinical studies.     

Alzheimer’s Disease-Associated Neurotoxic Peptide Amyloid-β Impairs Base Excision Repair in Human Neuroblastoma Cells

Alzheimer’s disease (AD) is the leading cause of dementia in developed countries. It is characterized by two major pathological hallmarks, one of which is the extracellular aggregation of the neurotoxic peptide amyloid-β (Aβ), which is known to generate oxidative stress. In this study, we showed that the presence of Aβ in a neuroblastoma cell line led to an increase in both nuclear and mitochondrial DNA damage. Unexpectedly, a concomitant decrease in basal level of base excision repair, a major route for repairing oxidative DNA damage, was observed at the levels of both gene expression and protein activity. Moreover, the addition of copper sulfate or hydrogen peroxide, used to mimic the oxidative stress observed in AD-affected brains, potentiates Aβ-mediated perturbation of DNA damage/repair systems in the “Aβ cell line”. Taken together, these findings indicate that Aβ could act as double-edged sword by both increasing oxidative nuclear/mitochondrial damage and preventing its repair. The synergistic effects of increased ROS production, accumulated DNA damage and impaired DNA repair could participate in, and partly explain, the massive loss of neurons observed in Alzheimer’s disease since both oxidative stress and DNA damage can trigger apoptosis.     

Mitochondrial Oxidative Stress—A Causative Factor and Therapeutic Target in Many Diseases

The excessive formation of reactive oxygen species (ROS) and impairment of defensive antioxidant systems leads to a condition known as oxidative stress. The main source of free radicals responsible for oxidative stress is mitochondrial respiration. The deleterious effects of ROS on cellular biomolecules, including DNA, is a well-known phenomenon that can disrupt mitochondrial function and contribute to cellular damage and death, and the subsequent development of various disease processes. In this review, we summarize the most important findings that implicated mitochondrial oxidative stress in a wide variety of pathologies from Alzheimer disease (AD) to autoimmune type 1 diabetes. This review also discusses attempts to affect oxidative stress as a therapeutic avenue.     

A glue method for fabricating membrane electrode assemblies for direct methanol fuel cells

We present a simple glue method for fabricating membrane electrode assemblies (MEA) for direct methanol fuel cells (DMFC). Rather than the conventional “dry” hot-pressing method that relies solely on hot-pressing at a high pressure and temperature to form a MEA, the “wet” method developed in this work introduces a binding agent, consisting of Nafion^® solution, between a polymer electrolyte membrane (PEM) and an anode/cathode. The introduced binding agent can provide a better adhesion and stronger binding force between a membrane and an electrode, thereby facilitating a better interfacial contact between the electrode and the Nafion^® membrane, which has been proved by scanning electron microscopy (SEM) analyses to the cross-sectional morphology of the MEA after long-term operation. The cell performance characterization showed the MEA fabricated by the glue method was more stable in cell performance than that fabricated by the conventional hot-pressing method. Cyclic voltammetry (CV) results also demonstrated the MEA fabricated by the glue method exhibited a higher electrochemical surface area (ESA) as a result of the improved interfacial contact between the Nafion^® membrane and the electrodes. Finally, the DMFC with the MEA fabricated by the glue method was characterized by the electrochemical impedance spectroscopy (EIS).     

Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis,DNA Repair,Inhibition of Cell Proliferation and Migration

Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2) called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE), which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix) polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen) and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair.     

Why Concurrent CDDP and Radiotherapy Has Synergistic Antitumor Effects: A Review of In Vitro Experimental and Clinical-Based Studies

Chemo-radiotherapy, which combines chemotherapy with radiotherapy, has been clinically practiced since the 1970s, and various anticancer drugs have been shown to have a synergistic effect when used in combination with radiotherapy. In particular, cisplatin (CDDP), which is often the cornerstone of multi-drug combination cancer therapies, is highly versatile and frequently used in combination with radiotherapy for the treatment of many cancers. Therefore, the mechanisms underlying the synergistic effect of CDDP and radiotherapy have been widely investigated, although no definitive conclusions have been reached. We present a review of the combined use of CDDP and radiotherapy, including the latest findings, and propose a mechanism that could explain their synergistic effects. Our hypothesis involves the concepts of overlap and complementation. “Overlap” refers to the overlapping reactions of CDDP and radiation-induced excessive oxidative loading, which lead to accumulating damage to cell components, mostly within the cytoplasm. “Complementation” refers to the complementary functions of CDDP and radiation that lead to DNA damage, primarily in the nucleus. In fact, the two concepts are inseparable, but conceptualizing them separately will help us understand the mechanism underlying the synergism between radiation therapy and other anticancer drugs, and help us to design future radiosensitizers.