Atherosclerosis and Coenzyme Q10.

Atherosclerosis is the most common cause of cardiac deaths worldwide. Classically, atherosclerosis has been explained as a simple arterial lipid deposition with concomitant loss of vascular elasticity. Eventually, this condition can lead to consequent blood flow reduction through the affected vessel. However, numerous studies have demonstrated that more factors than lipid accumulation are involved in arterial damage at the cellular level, such as inflammation, autophagy impairment, mitochondrial dysfunction, and/or free-radical overproduction. In order to consider the correction of all of these pathological changes, new approaches in atherosclerosis treatment are necessary. Ubiquinone or coenzyme Q10 is a multifunctional molecule that could theoretically revert most of the cellular alterations found in atherosclerosis, such as cholesterol biosynthesis dysregulation, impaired autophagy flux and mitochondrial dysfunction thanks to its redox and signaling properties. In this review, we will show the latest advances in the knowledge of the relationships between coenzyme Q10 and atherosclerosis. In addition, as atherosclerosis phenotype is closely related to aging, it is reasonable to believe that coenzyme Q10 supplementation could be beneficial for both conditions.

Two coffins and a funeral: early or late caspase activation determines two types of apoptosis induced by DNA damaging agents.

Cell cytoskeleton makes profound changes during apoptosis including the organization of an Apoptotic Microtubule Network (AMN). AMN forms a cortical structure which plays an important role in preserving plasma membrane integrity during apoptosis. Here, we examined the cytoskeleton rearrangements during apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. Using fixed and living cell imaging, we showed that CPT induced two dose- and cell cycle-dependent types of apoptosis characterized by different cytoskeleton reorganizations, time-dependent caspase activation and final apoptotic cell morphology. In the one referred as "slow" (~h) or round-shaped, apoptosis was characterized by a slow contraction of the actinomyosin ring and late caspase activation. In "slow" apoptosis the γ-tubulin complexes were not disorganized and microtubules were not depolymerized at early stages. In contrast, "fast" (~min) or irregular-shaped apoptosis was characterized by early caspase activation followed by full contraction of the actinomyosin ring. In fast apoptosis γ-tubulin complexes were disorganized and microtubules were initially depolymerized. However, after actinomyosin contraction, microtubules were reformed adopting a cortical but irregular disposition near plasma membrane. In addition to distinctive cytoskeleton reorganization kinetics, round and irregular-shaped apoptosis showed different biological properties with respect to AMN maintenance, plasma membrane integrity and phagocytes response. Our results suggest that the knowledge and modulation of the type of apoptosis promoted by genotoxic agents may be important for deciding a better therapeutic option and predicting the immune response in cancer treatment.

Dynamic Reorganization of the Cytoskeleton during Apoptosis: The Two Coffins Hypothesis.

During apoptosis, cells undergo characteristic morphological changes in which the cytoskeleton plays an active role. The cytoskeleton rearrangements have been mainly attributed to actinomyosin ring contraction, while microtubule and intermediate filaments are depolymerized at early stages of apoptosis. However, recent results have shown that microtubules are reorganized during the execution phase of apoptosis forming an apoptotic microtubule network (AMN). Evidence suggests that AMN is required to maintain plasma membrane integrity and cell morphology during the execution phase of apoptosis. The new "two coffins" hypothesis proposes that both AMN and apoptotic cells can adopt two morphological patterns, round or irregular, which result from different cytoskeleton kinetic reorganization during the execution phase of apoptosis induced by genotoxic agents. In addition, round and irregular-shaped apoptosis showed different biological properties with respect to AMN maintenance, plasma membrane integrity and phagocyte responses. These findings suggest that knowing the type of apoptosis may be important to predict how fast apoptotic cells undergo secondary necrosis and the subsequent immune response. From a pathological point of view, round-shaped apoptosis can be seen as a physiological and controlled type of apoptosis, while irregular-shaped apoptosis can be considered as a pathological type of cell death closer to necrosis.

Apoptotic cells subjected to cold/warming exposure disorganize apoptotic microtubule network and undergo secondary necrosis.

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 α-spectrin, paxilin, focal adhesion kinase 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.

Targeted delivery of pharmacological chaperones for Gaucher disease to macrophages by a mannosylated cyclodextrin carrier.

Gaucher disease (GD) is a rare monogenetic disorder leading to dysfunction of acid ß-glucosidase (ß-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 ß-cyclodextrin (ßCD) conjugate and the macrophage mannose receptor (MMR). First, PC candidates with appropriate relative avidities towards the ßCD 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 ßCD carrier was selectively recognized by mannose-specific lectins and that the corresponding PC:mannosylated ßCD 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.

Secondary coenzyme Q10 deficiency triggers mitochondria degradation by mitophagy in MELAS fibroblasts.

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.

Apoptotic microtubule network organization and maintenance depend on high cellular ATP levels and energized mitochondria.

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.

Pathophysiological characterization of MERRF patient-specific induced neurons generated by direct reprogramming.

Mitochondrial diseases are a group of rare heterogeneous genetic disorders caused by total or partial mitochondrial dysfunction. They can be caused by mutations in nuclear or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most common mitochondrial disorders caused by point mutations in mtDNA. It is mainly caused by the m.8344A > G mutation in the tRNALys (UUR) gene of mtDNA (MT-TK gene). This mutation affects the translation of mtDNA encoded proteins; therefore, the assembly of the electron transport chain (ETC) complexes is disrupted, leading to a reduced mitochondrial respiratory function. However, the molecular pathogenesis of MERRF syndrome remains poorly understood due to the lack of appropriate cell models, particularly in those cell types most affected in the disease such as neurons. Patient-specific induced neurons (iNs) are originated from dermal fibroblasts derived from different individuals carrying the particular mutation causing the disease. Therefore, patient-specific iNs can be used as an excellent cell model to elucidate the mechanisms underlying MERRF syndrome. Here we present for the first time the generation of iNs from MERRF dermal fibroblasts by direct reprograming, as well as a series of pathophysiological characterizations which can be used for testing the impact of a specific mtDNA mutation on neurons and screening for drugs that can correct the phenotype.

Pantothenate Rescues Iron Accumulation in Pantothenate Kinase-Associated Neurodegeneration Depending on the Type of Mutation.

Neurodegeneration with brain iron accumulation (NBIA) is a group of inherited neurologic disorders in which iron accumulates in the basal ganglia resulting in progressive dystonia, spasticity, parkinsonism, neuropsychiatric abnormalities, and optic atrophy or retinal degeneration. The most prevalent form of NBIA is pantothenate kinase-associated neurodegeneration (PKAN) associated with mutations in the gene of pantothenate kinase 2 (PANK2), which is essential for coenzyme A (CoA) synthesis. There is no cure for NBIA nor is there a standard course of treatment. In the current work, we describe that fibroblasts derived from patients harbouring PANK2 mutations can reproduce many of the cellular pathological alterations found in the disease, such as intracellular iron and lipofuscin accumulation, increased oxidative stress, and mitochondrial dysfunction. Furthermore, mutant fibroblasts showed a characteristic senescent morphology. Treatment with pantothenate, the PANK2 enzyme substrate, was able to correct all pathological alterations in responder mutant fibroblasts with residual PANK2 enzyme expression. However, pantothenate had no effect on mutant fibroblasts with truncated/incomplete protein expression. The positive effect of pantothenate in particular mutations was also confirmed in induced neurons obtained by direct reprograming of mutant fibroblasts. Our results suggest that pantothenate treatment can stabilize the expression levels of PANK2 in selected mutations. These results encourage us to propose our screening model as a quick and easy way to detect pantothenate-responder patients with PANK2 mutations. The existence of residual enzyme expression in some affected individuals raises the possibility of treatment using high dose of pantothenate.

Intracellular cholesterol accumulation and coenzyme Q10 deficiency in Familial Hypercholesterolemia.

Familial Hypercholesterolemia (FH) is an autosomal co-dominant genetic disorder characterized by elevated low-density lipoprotein (LDL) cholesterol levels and increased risk for premature cardiovascular disease. Here, we examined FH pathophysiology in skin fibroblasts derived from FH patients harboring heterozygous mutations in the LDL-receptor. Fibroblasts from FH patients showed a reduced LDL-uptake associated with increased intracellular cholesterol levels and coenzyme Q10 (CoQ10) deficiency, suggesting dysregulation of the mevalonate pathway. Secondary CoQ10 deficiency was associated with mitochondrial depolarization and mitophagy activation in FH fibroblasts. Persistent mitophagy altered autophagy flux and induced inflammasome activation accompanied by increased production of cytokines by mutant cells. All the pathological alterations in FH fibroblasts were also reproduced in a human endothelial cell line by LDL-receptor gene silencing. Both increased intracellular cholesterol and mitochondrial dysfunction in FH fibroblasts were partially restored by CoQ10 supplementation. Dysregulated mevalonate pathway in FH, including increased expression of cholesterogenic enzymes and decreased expression of CoQ10 biosynthetic enzymes, was also corrected by CoQ10 treatment. Reduced CoQ10 content and mitochondrial dysfunction may play an important role in the pathophysiology of early atherosclerosis in FH. The diagnosis of CoQ10 deficiency and mitochondrial impairment in FH patients may also be important to establish early treatment with CoQ10.

The Connections Among Autophagy, Inflammasome and Mitochondria.

BACKGROUND: The molecular crosstalk between inflammation and autophagy is an emerging field of research that is essential for the understanding of multicellular organism homeostasis and how these processes influence a variety of pathological conditions. OBJECTIVE: In this review, we briefly describe the relationship between autophagy and inflammasome activation. The central role that mitochondria play in both cellular processes is also discussed. CONCLUSION: Inflammasome and autophagy often modulate each other by common inhibitory mechanisms that are controlled by different input pathways. Thus, inflammasome components coordinate autophagy and autophagy regulates inflammasome activation, making the balance between both processes a fundamental player in cellular homeostasis.

Fluorinated Chaperone-ß-Cyclodextrin Formulations for ß-Glucocerebrosidase Activity Enhancement in Neuronopathic Gaucher Disease.

Amphiphilic glycomimetics encompassing a rigid, undistortable nortropane skeleton based on 1,6-anhydro-l-idonojirimycin and a polyfluorinated antenna, when formulated as the corresponding inclusion complexes with ß-cyclodextrin (ßCD), have been shown to behave as pharmacological chaperones (PCs) that efficiently rescue lysosomal ß-glucocerebrosidase mutants associated with the neuronopathic variants of Gaucher disease (GD), including the highly refractory L444P/L444P and L444P/P415R single nucleotide polymorphs, in patient fibroblasts. The body of work here presented includes the design criteria for the PC prototype, the synthesis of a series of candidates, the characterization of the PC:ßCD complexes, the determination of the selectivity profiles toward a panel of commercial and human lysosomal glycosidases, the evaluation of the chaperoning activity in type 1 (non-neuronopathic), type 2 (acute neuronopathic), and type 3 (adult neuronopathic) GD fibroblasts, the confirmation of the rescuing mechanism by immunolabeling, and the analysis of the PC:GCase binding mode by docking experiments.

Coenzyme Q10 partially restores pathological alterations in a macrophage model of Gaucher disease.

BACKGROUND: Gaucher disease (GD) is caused by mutations in the GBA1 gene which encodes lysosomal ß-glucocerebrosidase (GCase). In GD, partial or complete loss of GCase activity causes the accumulation of the glycolipids glucosylceramide (GlcCer) and glucosylsphingosine in the lysosomes of macrophages. In this manuscript, we investigated the effects of glycolipids accumulation on lysosomal and mitochondrial function, inflammasome activation and efferocytosis capacity in a THP-1 macrophage model of Gaucher disease. In addition, the beneficial effects of coenzyme Q10 (CoQ) supplementation on cellular alterations were evaluated. Chemically-induced Gaucher macrophages were developed by differentiateing THP-1 monocytes to macrophages by treatment with phorbol 12-myristate 13-acetate (PMA) and then inhibiting intracellular GCase with conduritol B-epoxide (CBE), a specific irreversible inhibitor of GCase activity, and supplementing the medium with exogenous GlcCer. This cell model accumulated up to 16-fold more GlcCer compared with control THP-1 cells. RESULTS: Chemically-induced Gaucher macrophages showed impaired autophagy flux associated with mitochondrial dysfunction and increased oxidative stress, inflammasome activation and impaired efferocytosis. All abnormalities were partially restored by supplementation with CoQ. CONCLUSION: These data suggest that targeting mitochondria function and oxidative stress by CoQ can ameliorate the pathological phenotype of Gaucher cells. Chemically-induced Gaucher macrophages provide cellular models that can be used to investigate disease pathogenesis and explore new therapeutics for GD.

Mitochondrial Dysfunction in Lysosomal Storage Disorders.

Lysosomal storage diseases (LSDs) describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS), where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm), diminished ATP production and increased generation of reactive oxygen species (ROS). Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD), the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme ß-glucocerebrosidase (GCase). Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph) in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs.

AMPK Regulation of Cell Growth, Apoptosis, Autophagy, and Bioenergetics.

In eukaryotic cells, AMP-activated protein kinase (AMPK) generally promotes catabolic pathways that produce ATP and at the same time inhibits anabolic pathways involved in different processes that consume ATP. As an energy sensor, AMPK is involved in the main cellular functions implicated in cell fate, such as cell growth and autophagy.Recently, AMPK has been connected with apoptosis regulation, although the molecular mechanism by which AMPK induces and/or inhibits cell death is not clear.This chapter reviews the essential role of AMPK in signaling pathways that respond to cellular stress and damage, highlighting the complex and reciprocal regulation between AMPK and their targets and effectors. The therapeutic implications of the role of AMPK in different pathologies such as diabetes, cancer, or mitochondrial dysfunctions are still controversial, and it is necessary to further investigate the molecular mechanisms underlying AMPK activation.

Targeting autophagy and mitophagy for mitochondrial diseases treatment.

INTRODUCTION: Mitochondrial diseases are a group of rare genetic diseases with complex and heterogeneous origins which manifest a great variety of phenotypes. Disruption of the oxidative phosphorylation system is the main cause of pathogenicity in mitochondrial diseases since it causes accumulation of reactive oxygen species (ROS) and ATP depletion. AREAS COVERED: Current evidences support the main protective role of autophagy and mitophagy in mitochondrial diseases and other diseases associated with mitochondrial dysfunction. EXPERT OPINION: The use of autophagy and/or mitophagy inducers may allow a novel strategy for improving mitochondrial function for both mitochondrial diseases and other diseases with altered mitochondrial metabolism. However, a deeper investigation of the molecular mechanisms behind mitophagy and mitochondrial biogenesis is needed in order to safely modulate these processes. In the coming years, we will also see an increase in awareness of mitochondrial dynamics modulation that will allow the therapeutic use of new drugs for improving mitochondrial function in a great variety of mitochondrial disorders.

AMPK As A Target in Rare Diseases.

The AMP-activated protein kinase (AMPK) has emerged as an important sensor of signals that control cellular energy balance in all eukaryotes. AMPK is also involved in fatty acid oxidation, glucose transport, antioxidant defense, mitochondrial biogenesis and the modulation of inflammatory processes. The numerous roles of AMPK in cell physiological and pathological states justified the notable increase in the number of publications in previous years, with almost 1500 scientific articles relative to this kinase in 2014. Due to its role in maintaining energy balance, a dysfunction in AMPK signalling pathway may result in perturbations at the systemic level that contribute to the development of many disease conditions. Among them, more than 7000 poorly-known rare diseases are particularly of social and scientific interest because they are usually chronically debilitating or even lifethreatening and lack effective and safe treatment. Several authors have demonstrated AMPK alterations and the beneficial effect of treatments with drugs regulating AMPK activity in some of these low prevalence pathologies. Among these rare diseases in which AMPK can play an important pathological role are mitochondrial disorders, muscular dystrophies, cardiovascular diseases, neurodegenerative pathologies, or even some types of cancer for the importance of AMPK as a suppressor of cell proliferation. This review focuses on current knowledge about the pathophysiological roles of AMPK and future approaches as therapeutic targeting in rare diseases.

Mitochondrial Dynamics in Mitochondrial Diseases.

Mitochondria are very versatile organelles in continuous fusion and fission processes in response to various cellular signals. Mitochondrial dynamics, including mitochondrial fission/fusion, movements and turnover, are essential for the mitochondrial network quality control. Alterations in mitochondrial dynamics can cause neuropathies such as Charcot-Marie-Tooth disease in which mitochondrial fusion and transport are impaired, or dominant optic atrophy which is caused by a reduced mitochondrial fusion. On the other hand, mitochondrial dysfunction in primary mitochondrial diseases promotes reactive oxygen species production that impairs its own function and dynamics, causing a continuous vicious cycle that aggravates the pathological phenotype. Mitochondrial dynamics provides a new way to understand the pathophysiology of mitochondrial disorders and other diseases related to mitochondria dysfunction such as diabetes, heart failure, or Hungtinton's disease. The knowledge about mitochondrial dynamics also offers new therapeutics targets in mitochondrial diseases.

Amitriptyline induces mitophagy that precedes apoptosis in human HepG2 cells.

Systemic treatments for hepatocellular carcinoma (HCC) have been largely unsuccessful. This study investigated the antitumoral activity of Amitriptyline, a tricyclic antidepressant, in hepatoma cells. Amitriptyline-induced toxicity involved early mitophagy activation that subsequently switched to apoptosis. Amitriptyline induced mitochondria dysfunction and oxidative stress in HepG2 cells. Amitriptyline specifically inhibited mitochondrial complex III activity that is associated with decreased mitochondrial membrane potential (∆Ψm) and increased reactive oxygen species (ROS) production. Transmission electron microscopy (TEM) studies revealed structurally abnormal mitochondria that were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitophagy activation, fluorescence microscopy analysis showed mitochondrial Parkin recruitment and colocalization of mitochondria with autophagosome protein markers. Pharmacological or genetic inhibition of autophagy exacerbated the deleterious effects of Amitriptyline on hepatoma cells and led to increased apoptosis. These results suggest that mitophagy acts as an initial adaptive mechanism of cell survival. However persistent mitochondrial damage induced extensive and lethal mitophagy, autophagy stress and autophagolysome permeabilization leading eventually to cell death by apoptosis. Amitriptyline also induced cell death in hepatoma cells lines with mutated p53 and non-sense p53 mutation. Our results support the hypothesis that Amitriptyline-induced mitochondrial dysfunction can be a useful therapeutic strategy for HCC treatment, especially in tumors showing p53 mutations and/or resistant to genotoxic treatments.