[Show abstract][Hide abstract] ABSTRACT: Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. We were able by using a cocktail composed of taxol (a microtubule stabilizer), Zn2+ (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant) to stabilize H460 apoptotic cells in cell cultures for at least 72 hours preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cells characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.
Cell Death & Disease 07/2014; · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath the plasma membrane which plays a critical role in preserving cell morphology and plasma membrane integrity. The aim of this study was to examine the effect of cold/warming exposure on apoptotic microtubules and plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptotic H460 cells that cold/warming exposure disorganized apoptotic microtubules and allowed the access of active caspases to the cellular cortex and the cleavage of essential proteins in the preservation of plasma membrane permeability. Cleavage of cellular cortex and plasma membrane proteins, such as -spectrin, paxilin, Focal Adhesion Kinase (FAK) and calcium ATPase pump (PMCA-4) involved in cell calcium extrusion resulted in increased plasma permeability and calcium overload leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the addition of the pan-caspase inhibitor z-VAD during cold/warming exposure that induces AMN depolymerization avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Likewise, apoptotic microtubules stabilization by taxol during cold/warming exposure also prevented cellular cortex and plasma membrane protein cleavage and secondary necrosis. Furthermore, microtubules stabilization or caspase inhibition during cold/warming exposure was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages.
These results indicate that cold/warming exposure of apoptotic cells induces secondary necrosis which can be prevented by both, microtubule stabilization or caspase inhibition.
[Show abstract][Hide abstract] ABSTRACT: Gaucher disease (GD) is a rare monogenetic disorder leading to dysfunction of acid β-glucosidase (β-glucocerebrosidase; GCase) and accumulation of glucosylceramide in lysosomes, especially in macrophages (Gaucher cells). Many of the mutations at the origin of GD do not impair the catalytic activity of GCase, but cause misfolding and subsequent degradation by the quality control system at the endoplasmic reticulum. Pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the endogenous mutant enzyme represent promising alternatives to the currently available enzyme replacement and substrate reduction therapies (ERT and SRT, respectively), but unfavorable biodistribution and potential side-effects remain important issues. We have now designed a strategy to enhance the controlled delivery of PCs to macrophages that exploit the formation of ternary complexes between the PC, a trivalent mannosylated β-cyclodextrin (βCD) conjugate and the macrophage mannose receptor (MMR). First, PC candidates with appropriate relative avidities towards the βCD cavity and the GCase active site were selected to ensure efficient transfer of the PC cargo from the host to the GCase active site. Control experiments confirmed that the βCD carrier was selectively recognized by mannose-specific lectins and that the corresponding PC:mannosylated βCD supramolecular complex retained both the chaperoning activity, as confirmed in human GD fibroblasts, and the MMR binding ability. Finally, fluorescence microscopy techniques proved targeting and cellular uptake of the PC-loaded system in macrophages. Altogether, the results support that combined cyclodextrin encapsulation and glycotargeting may improve the efficacy of PCs for GD.
[Show abstract][Hide abstract] ABSTRACT: Camptothecin (CPT; (S)-(+)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-
b]quinoline-3,14-(4H,12H)-dione) is a highly cytotoxic natural alkaloid that has not yet
found use as chemotherapeutic agent due to its poor water-solubility and chemical
instability and, as a consequence, no effective administration means have been
designed. In this work, camptothecin has been successfully loaded into iron oxide
superparamagnetic nanoparticles with average size 14 nm. It was found that surface
modification of the nanoparticles by polyethylene glycol enables loading a large
amount of camptothecin. While the unloaded nanoparticles do not induce apoptosis
in H460 lung cancer cell line, the camptothecin-loaded nanoparticle formulations
exhibit remarkable pro-apoptotic activity. These results indicate that camptothecin
retains its biological activity after loading onto the magnetic nanoparticles. The
proposed materials represent novel materials based on naturally occurring bioactive
molecules loaded onto nanoparticles to be used as chemotherapeutic formulations.
The procedure seems apt to be extended to other active molecules extracted from
natural products. In addition, these materials offer the potential of being further
implemented for combined imaging and therapeutics, as magnetic nanoparticles are
known to be multifunctional tools for biomedicine.
Beilstein Journal of Nanotechnology 01/2014; · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coenzyme Q₁₀ (Co Q₁₀) or ubiquinone was known for its key role in mitochondrial bioenergetics as electron and proton carrier; later studies demonstrated its presence in other cellular membranes and in blood plasma, and extensively investigated its antioxidant role. These two functions constitute the basis for supporting the clinical indication of Co Q₁₀. Furthermore, recent data indicate that Co Q₁₀ affects expression of genes involved in human cell signalling, metabolism and transport and some of the effects of Co Q₁₀ supplementation may be due to this property. Co Q₁₀ deficiencies are due to autosomal recessive mutations, mitochondrial diseases, ageing-related oxidative stress and carcinogenesis processes, and also a secondary effect of statin treatment. Many neurodegenerative disorders, diabetes, cancer, fibromyalgia, muscular and cardiovascular diseases have been associated with low Co Q₁₀ levels. Co Q₁₀ treatment does not cause serious adverse effects in humans and new formulations have been developed that increase Co Q₁₀ absorption and tissue distribution. Oral Co Q₁₀ treatment is a frequent mitochondrial energizer and antioxidant strategy in many diseases that may provide a significant symptomatic benefit.
Frontiers in Bioscience 01/2014; 19:619-33. · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For a number of years, coenzyme Q10 (CoQ10) was known for its key role in mitochondrial bioenergetics; later studies demonstrated its presence in other subcellular fractions and in blood plasma, and extensively investigated its antioxidant role. These two functions constitute the basis for supporting the clinical use of CoQ10. Also at the inner mitochondrial membrane level, CoQ10 is recognized as an obligatory co-factor for the function of uncoupling proteins and a modulator of the mitochondrial transition pore. Furthermore, recent data indicate that CoQ10 affects expression of genes involved in human cell signalling, metabolism, and transport and some of the effects of CoQ10 supplementation may be due to this property.
CoQ10 deficiencies are due to autosomal recessive mutations, mitochondrial diseases, ageing-related oxidative stress and carcinogenesis processes, and also statin treatment. Many neurodegenerative disorders, diabetes, cancer and muscular and cardiovascular diseases have been associated with low CoQ10 levels, as well as different ataxias and encephalomyopathies.
CoQ10 treatment does not cause serious adverse effects in humans and new formulations have been developed that increase CoQ10 absorption and tissue distribution. Oral CoQ10 is a frequent antioxidant strategy in many diseases that may provide a significant symptomatic benefit.
[Show abstract][Hide abstract] ABSTRACT: Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na/Ca exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na/K pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis.
Cell Death & Disease 01/2013; 4:e527. · 6.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Silver nanoparticles are emerging as a powerful tool in bioimaging applications owing to their unique plasmonic properties i.e., extremely high molar extinction coefficients, resonant Rayleigh scattering and enhanced local electromagnetic fields. Through the optimization of these properties, by controlling composition, size, shape, and interparticle spacing of nanoparticles and their assemblies, highly enhanced local electromagnetic fields in the vicinity of nanoparticles are achievable giving rise to IR, Raman and fluorescence surface enhanced spectroscopies (SEIRS, SERS and MEF, respectively).
Microscopy and Microanalysis 08/2012; 18(S5):53-54. · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is a growing interest in nanoparticles as carriers of chemotherapeutic agents in order to improve their administration and minimize their side effects. Despite the fact that silver nanoparticles can be conjugated to therapeutic agents, offering additionally advantages due their unique and tunable optical properties, few examples have been described yet.
Microscopy and Microanalysis 08/2012; 18(S5):55-56. · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND PURPOSE MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. EXPERIMENTAL APPROACH We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. KEY RESULTS According to our results, supplementation with riboflavin or coenzyme Q(10) effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. CONCLUSIONS AND IMPLICATIONS Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment.
British Journal of Pharmacology 07/2012; 167(6):1311-1328. · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial DNA mutations are an important cause of human disease for which there is no effective treatment. Myoclonic epilepsy with ragged-red fibers (MERRF) is a mitochondrial disease usually caused by point mutations in transfer RNA genes encoded by mitochondrial DNA. The most common mutation associated with MERRF syndrome, m.8344A > G in the gene MT-TK, which encodes transfer RNA(Lysine), affects the translation of all mitochondrial DNA encoded proteins. This impairs the assembly of the electron transport chain complexes leading to decreased mitochondrial respiratory function. Here we report on how this mutation affects mitochondrial function in primary fibroblast cultures established from patients harboring the A8344G mutation. Coenzyme Q₁₀ (CoQ) levels, as well as mitochondrial respiratory chain activity, and mitochondrial protein expression levels were significantly decreased in MERRF fibroblasts. Mitotracker staining and imaging analysis of individual mitochondria indicated the presence of small, rounded, depolarized mitochondria in MERRF fibroblasts. Mitochondrial dysfunction was associated with increased oxidative stress and increased degradation of impaired mitochondria by mitophagy. Transmitochondrial cybrids harboring the A8344G mutation also showed CoQ deficiency, mitochondrial dysfunction, and increased mitophagy activity. All these abnormalities in patient-derived fibroblasts and cybrids were partially restored by CoQ supplementation, indicating that these cell culture models may be suitable for screening and validation of novel drug candidates for MERRF disease.
Journal of the American Society for Experimental NeuroTherapeutics 02/2012; 9(2):446-63. · 5.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mtDNA. Here, we report on how this mutation affects mitochondrial function in primary fibroblast cultures established from 2 patients with MELAS who harbored the A3243G mutation. Both mitochondrial respiratory chain enzyme activities and coenzyme Q(10) (CoQ) levels were significantly decreased in MELAS fibroblasts. A similar decrease in mitochondrial membrane potential was found in intact MELAS fibroblasts. Mitochondrial dysfunction was associated with increased oxidative stress and the activation of mitochondrial permeability transition (MPT), which triggered the degradation of impaired mitochondria. Furthermore, we found defective autophagosome elimination in MELAS fibroblasts. Electron and fluorescence microscopy studies confirmed a massive degradation of mitochondria and accumulation of autophagosomes, suggesting mitophagy activation and deficient autophagic flux. Transmitochondrial cybrids harboring the A3243G mutation also showed CoQ deficiency and increased autophagy activity. All these abnormalities were partially restored by CoQ supplementation. Autophagy in MELAS fibroblasts was also abolished by treatment with antioxidants or cyclosporine, suggesting that both reactive oxygen species and MPT participate in this process. Furthermore, prevention of autophagy in MELAS fibroblasts resulted in apoptotic cell death, suggesting a protective role of autophagy in MELAS fibroblasts.
The FASEB Journal 05/2011; 25(8):2669-87. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in "reverse" mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.