Mitochondria and Reactive Oxygen Species: The Therapeutic Balance of Powers for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle-wasting disorder that leads to rapid loss of mobility and premature death. The absence of functional dystrophin in DMD patients reduces sarcolemma stiffness and increases contraction damage, triggering a cascade of events leading to muscle cell degeneration, chronic inflammation, and deposition of fibrotic and adipose tissue. Efforts in the last decade have led to the clinical approval of novel drugs for DMD that aim to restore dystrophin function. However, combination therapies able to restore dystrophin expression and target the myriad of cellular events found impaired in dystrophic muscle are desirable. Muscles are higher energy consumers susceptible to mitochondrial defects. Mitochondria generate a significant source of reactive oxygen species (ROS), and they are, in turn, sensitive to proper redox balance. In both DMD patients and animal models there is compelling evidence that mitochondrial impairments have a key role in the failure of energy homeostasis. Here, we highlighted the main aspects of mitochondrial dysfunction and oxidative stress in DMD and discussed the recent findings linked to mitochondria/ROS-targeted molecules as a therapeutic approach. In this respect, dual targeting of both mitochondria and redox homeostasis emerges as a potential clinical option in DMD.

Mecp2 knock-out astrocytes affect synaptogenesis by interleukin 6 dependent mechanisms.

Synaptic abnormalities are a hallmark of several neurological diseases, and clarification of the underlying mechanisms represents a crucial step toward the development of therapeutic strategies. Rett syndrome (RTT) is a rare neurodevelopmental disorder, mainly affecting females, caused by mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, leading to a deep derangement of synaptic connectivity. Although initial studies supported the exclusive involvement of neurons, recent data have highlighted the pivotal contribution of astrocytes in RTT pathogenesis through non-cell autonomous mechanisms. Since astrocytes regulate synapse formation and functionality by releasing multiple molecules, we investigated the influence of soluble factors secreted by Mecp2 knock-out (KO) astrocytes on synapses. We found that Mecp2 deficiency in astrocytes negatively affects their ability to support synaptogenesis by releasing synaptotoxic molecules. Notably, neuronal inputs from a dysfunctional astrocyte-neuron crosstalk lead KO astrocytes to aberrantly express IL-6, and blocking IL-6 activity prevents synaptic alterations.

Neuroprotective role of plumbagin on eye damage induced by high-sucrose diet in adult fruit fly Drosophila melanogaster.

The natural compound plumbagin has a wide range of pharmacological and potential therapeutic activities, although its role in neuroretina degeneration is unknown. Here we evaluated the effects of plumbagin on retina homeostasis of the fruit fly Drosophila melanogaster fed with high glucose diet, a model of hyperglycemia-induced eye impairment to study the pathophysiology of diabetic retinopathy at the early stages. To this aim, the visual system of flies orally administered with plumbagin has been analyzed at structural, functional, and molecular/cellular level as for instance neuronal apoptosis/autophagy dysregulation and oxidative stress-related signals. Our results demonstrated that plumbagin ameliorates the visual performance of hyperglycemic flies. Drosophila eye-structure, clearly altered by hyperglycemia, i.e. defects of the pattern of ommatidia, irregular rhabdomeres, vacuoles, damaged mitochondria, and abnormal phototransduction units were rescued, at least in part, by plumbagin. In addition, it reactivated autophagy, decreased the presence of cell death/apoptotic features, and exerted antioxidant effects in the retina. In terms of mechanisms favoring death/survival ratio, Nrf2 signaling activation may be one of the strategies by which plumbagin reduced redox unbalance mainly increasing the levels of glutathione-S-transferase. Likewise, plumbagin may act additively and/or synergistically inhibiting the mitochondrial-endoplasmic reticulum stress and unfolded protein response pathways, which prevented neuronal impairment and eye damage induced by reactive oxygen species. These results provide an avenue for further studies, which may be helpful to develop novel therapeutic candidates and drug targets against eye neurotoxicity by high glucose, a key aspect in retinal complications of diabetes.

Serum starvation affects mitochondrial metabolism of adipose-derived stem/stromal cells.

BACKGROUND AIMS: A large part of mesenchymal stromal cell (MSC) regenerative and immunomodulatory action is mediated by paracrine signaling. Hence, an increasing body of evidence acknowledges the potential of MSC secretome in a variety of preclinical and clinical scenarios. Mid-term serum deprivation is a common approach in the pipeline of MSC secretome production. Nevertheless, up to now, little is known about the impact of this procedure on the metabolic status of donor cells. METHODS: Here, through untargeted differential metabolomics, we revealed an impairment of mitochondrial metabolism in adipose-derived MSCs exposed for 72 h to serum deprivation. RESULTS: This evidence was further confirmed by the significant accumulation of reactive oxygen species and the reduction of succinate dehydrogenase activity. Probably as a repair mechanism, an upregulation of mitochondrial superoxide dismutase was also induced. CONCLUSIONS: Of note, the analysis of mitochondrial functionality indicated that, despite a significant reduction of basal respiration and ATP production, serum-starved MSCs still responded to changes in energy demand. This metabolic phenotype correlates with the obtained evidence of mitochondrial elongation and branching upon starvation.

RACK1 is evolutionary conserved in satellite stem cell activation and adult skeletal muscle regeneration.

Skeletal muscle growth and regeneration involves the activity of resident adult stem cells, namely satellite cells (SC). Despite numerous mechanisms have been described, different signals are emerging as relevant in SC homeostasis. Here we demonstrated that the Receptor for Activated C-Kinase 1 (RACK1) is important in SC function. RACK1 was expressed transiently in the skeletal muscle of post-natal mice, being abundant in the early phase of muscle growth and almost disappearing in adult mature fibers. The presence of RACK1 in interstitial SC was also detected. After acute injury in muscle of both mouse and the fruit fly Drosophila melanogaster (used as alternative in vivo model) we found that RACK1 accumulated in regenerating fibers while it declined with the progression of repair process. To note, RACK1 also localized in the active SC that populate recovering tissue. The dynamics of RACK1 levels in isolated adult SC of mice, i.e., progressively high during differentiation and low compared to proliferating conditions, and RACK1 silencing indicated that RACK1 promotes both the formation of myotubes and the accretion of nascent myotubes. In Drosophila with depleted RACK1 in all muscle cells or, specifically, in SC lineage we observed a delayed recovery of skeletal muscle after physical damage as well as the low presence of active SC in the wound area. Our results also suggest the coupling of RACK1 to muscle unfolded protein response during SC activation. Collectively, we provided the first evidence that transient levels of the evolutionarily conserved factor RACK1 are critical for adult SC activation and proper skeletal muscle regeneration, favoring the efficient progression of SC from a committed to a fully differentiated state.

Evidence of a dual mechanism of action underlying the anti-proliferative and cytotoxic effects of ammonium-alkyloxy-stilbene-based α7- and α9-nicotinic ligands on glioblastoma cells.

Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.

Acid Sphingomyelinase Controls Early Phases of Skeletal Muscle Regeneration by Shaping the Macrophage Phenotype.

Skeletal muscle regeneration is a complex process involving crosstalk between immune cells and myogenic precursor cells, i.e., satellite cells. In this scenario, macrophage recruitment in damaged muscles is a mandatory step for tissue repair since pro-inflammatory M1 macrophages promote the activation of satellite cells, stimulating their proliferation and then, after switching into anti-inflammatory M2 macrophages, they prompt satellite cells' differentiation into myotubes and resolve inflammation. Here, we show that acid sphingomyelinase (ASMase), a key enzyme in sphingolipid metabolism, is activated after skeletal muscle injury induced in vivo by the injection of cardiotoxin. ASMase ablation shortens the early phases of skeletal muscle regeneration without affecting satellite cell behavior. Of interest, ASMase regulates the balance between M1 and M2 macrophages in the injured muscles so that the absence of the enzyme reduces inflammation. The analysis of macrophage populations indicates that these events depend on the altered polarization of M1 macrophages towards an M2 phenotype. Our results unravel a novel role of ASMase in regulating immune response during muscle regeneration/repair and suggest ASMase as a supplemental therapeutic target in conditions of redundant inflammation that impairs muscle recovery.

Nutraceutical Strategy to Counteract Eye Neurodegeneration and Oxidative Stress in Drosophila melanogaster Fed with High-Sugar Diet.

Aberrant production of reactive oxygen species (ROS) is a common feature of damaged retinal neurons in diabetic retinopathy, and antioxidants may exert both preventive and therapeutic action. To evaluate the beneficial and antioxidant properties of food supplementation with Lisosan G, a powder of bran and germ of grain (Triticum aestivum) obtained by fermentation with selected lactobacillus and natural yeast strains, we used an in vivo model of hyperglycemia-induced retinal damage, the fruit fly Drosophila melanogaster fed with high-sucrose diet. Lisosan G positively affected the visual system of hyperglycemic flies at structural/functional level, decreased apoptosis, and reactivated protective autophagy at the retina internal network. Also, in high sucrose-fed Drosophila, Lisosan G reduced the levels of brain ROS and retina peroxynitrite. The analysis of oxidative stress-related metabolites suggested 7,8-dihydrofolate, uric acid, dihydroorotate, γ-L-glutamyl-L-cysteine, allantoin, cysteinyl-glycine, and quinolate as key mediators of Lisosan G-induced inhibition of neuronal ROS, along with the upregulation of glutathione system. Of note, Lisosan G may impact oxidative stress and the ensuing retinal cell death, also independently from autophagy, although the autophagy-ROS cross-talk is critical. This study demonstrated that the continuous supplementation with the alimentary integrator Lisosan G exerts a robust and multifaceted antioxidant effect on retinal neurons, thus providing efficacious neuroprotection of hyperglycemic eye.

Targeting HDAC8 to ameliorate skeletal muscle differentiation in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle degeneration and currently there are few therapeutic options. The identification of new drug targets and their validation in model systems of DMD could be a promising approach to make progress in finding new treatments for this lethal disease. Histone deacetylases (HDACs) play key roles in myogenesis and the therapeutic approach targeting HDACs in DMD is in an advanced phase of clinical trial. Here, we show that the expression of HDAC8, one of the members of the HDAC family, is increased in DMD patients and dystrophic zebrafish. The selective inhibition of HDAC8 with the PCI-34051 inhibitor rescues skeletal muscle defects, similarly to the treatment with the pan-HDAC inhibitor Givinostat. Through acetylation profile of zebrafish with HDAC8 dysregulation, we identified new HDAC8 targets involved in cytoskeleton organization such as tubulin that, when acetylated, is a marker of stable microtubules. Our work provides evidence of HDAC8 overexpression in DMD patients and zebrafish and supports its specific inhibition as a new valuable therapeutic approach in the treatment of this pathology.

Givinostat as metabolic enhancer reverting mitochondrial biogenesis deficit in Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a rare disorder characterized by progressive muscle wasting, weakness, and premature death. Remarkable progress has been made in genetic approaches, restoring dystrophin, or its function. However, the targeting of secondary pathological mechanisms, such as increasing muscle blood flow or stopping fibrosis, remains important to improve the therapeutic benefits, that depend on tackling both the genetic disease and the downstream consequences. Mitochondrial dysfunctions are one of the earliest deficits in DMD, arise from multiple cellular stressors and result in less than 50% of ATP content in dystrophic muscles. Here we establish that there are two temporally distinct phases of mitochondrial damage with depletion of mitochondrial mass at early stages and an accumulation of dysfunctional mitochondria at later stages, leading to a different oxidative fibers pattern, in young and adult mdx mice. We also observe a progressive mitochondrial biogenesis impairment associated with increased deacetylation of peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) promoter. Such histone deacetylation is inhibited by givinostat that positively modifies the epigenetic profile of PGC-1α promoter, sustaining mitochondrial biogenesis and oxidative fiber type switch. We, therefore, demonstrate that givinostat exerts relevant effects at mitochondrial level, acting as a metabolic remodeling agent capable of efficiently promoting mitochondrial biogenesis in dystrophic muscle.

Retinal damage in a new model of hyperglycemia induced by high-sucrose diets.

Loss of retinal neurons may precede clinical signs of diabetic retinopathy (DR). We studied for the first time the effects of hyperglycemia on the visual system of the fruit fly Drosophila melanogaster to characterize a model for glucose-induced retinal neurodegeneration, thus complementing more traditional vertebrate systems. Adult flies were fed with increased high-sucrose regimens which did not modify the locomotion ability, muscle phenotype and mobility after 10 days. The increased availability of dietary sucrose induced hyperglycemia and phosphorylation of Akt in fat tissue, without significant effects on adult growth and viability, consistent with the early phase of insulin signaling and a low impact on the overall metabolic profile of flies at short term. Noteworthy, high-sucrose diets significantly decreased Drosophila responsiveness to the light as a consequence of vision defects. Hyperglycemia did not alter the gross anatomical architecture of the external eye phenotype although a progressive damage of photosensitive units was observed. Appreciable levels of cleaved caspase 3 and nitrotyrosine were detected in the internal retina network as well as punctate staining of Light-Chain 3 and p62, and accumulated autophagosomes, indicating apoptotic features, peroxynitrite formation and autophagy turnover defects. In summary, our results in Drosophila support the view that hyperglycemia induced by high-sucrose diets lead to eye defects, apoptosis/autophagy dysregulation, oxidative stress, and visual dysfunctions which are evolutionarily conserved, thus offering a meaningful opportunity of using a simple in vivo model to study the pathophysiology of neuroretinal alterations that develop in patients at the early stages of DR.

In vivo magnetic resonance spectroscopy in the brain of Cdkl5 null mice reveals a metabolic profile indicative of mitochondrial dysfunctions.

Mutations in the X-linked CDKL5 gene cause CDKL5 deficiency disorder (CDD), a severe neurodevelopmental condition mainly characterized by infantile epileptic encephalopathy, intellectual disability, and autistic features. The molecular mechanisms underlying the clinical symptoms remain largely unknown and the identification of reliable biomarkers in animal models will certainly contribute to increase our comprehension of CDD as well as to assess the efficacy of therapeutic strategies. Here, we used different Magnetic Resonance (MR) methods to disclose structural, functional, or metabolic signatures of Cdkl5 deficiency in the brain of adult mice. We found that loss of Cdkl5 does not cause cerebral atrophy but affects distinct brain areas, particularly the hippocampus. By in vivo proton-MR spectroscopy (MRS), we revealed in the Cdkl5 null brain a metabolic dysregulation indicative of mitochondrial dysfunctions. Accordingly, we unveiled a significant reduction in ATP levels and a decrease in the expression of complex IV of mitochondrial electron transport chain. Conversely, the number of mitochondria appeared preserved. Importantly, we reported a significant defect in the activation of one of the major regulators of cellular energy balance, the adenosine monophosphate-activated protein kinase (AMPK), that might contribute to the observed metabolic impairment and become an interesting therapeutic target for future preclinical trials. In conclusion, MRS revealed in the Cdkl5 null brain the presence of a metabolic dysregulation suggestive of a mitochondrial dysfunction that permitted to foster our comprehension of Cdkl5 deficiency and brought our interest towards targeting mitochondria as therapeutic strategy for CDD.

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy.

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.

Defects of full-length dystrophin trigger retinal neuron damage and synapse alterations by disrupting functional autophagy.

Dystrophin (dys) mutations predispose Duchenne muscular disease (DMD) patients to brain and retinal complications. Although different dys variants, including long dys products, are expressed in the retina, their function is largely unknown. We investigated the putative role of full-length dystrophin in the homeostasis of neuro-retina and its impact on synapsis stabilization and cell fate. Retinas of mdx mice, the most used DMD model which does not express the 427-KDa dys protein (Dp427), showed overlapped cell death and impaired autophagy. Apoptotic neurons in the outer plexiform/inner nuclear layer and the ganglion cell layer had an impaired autophagy with accumulated autophagosomes. The autophagy dysfunction localized at photoreceptor axonal terminals and bipolar, amacrine, and ganglion cells. The absence of Dp427 does not cause a severe phenotype but alters the neuronal architecture, compromising mainly the pre-synaptic photoreceptor terminals and their post-synaptic sites. The analysis of two dystrophic mutants of the fruit fly Drosophila melanogaster, the homozygous DysE17 and DysEP3397, lacking functional large-isoforms of dystrophin-like protein, revealed rhabdomere degeneration. Structural damages were evident in the internal network of retina/lamina where photoreceptors make the first synapse. Both accumulated autophagosomes and apoptotic features were detected and the visual system was functionally impaired. The reactivation of the autophagosome turnover by rapamycin prevented neuronal cell death and structural changes of mutant flies and, of interest, sustained autophagy ameliorated their response to light. Overall, these findings indicate that functional full-length dystrophin is required for synapsis stabilization and neuronal survival of the retina, allowing also proper autophagy as a prerequisite for physiological cell fate and visual properties.

Autophagy in the Regulation of Tissue Differentiation and Homeostasis.

Autophagy is a constitutive pathway that allows the lysosomal degradation of damaged components. This conserved process is essential for metabolic plasticity and tissue homeostasis and is crucial for mammalian post-mitotic cells. Autophagy also controls stem cell fate and defective autophagy is involved in many pathophysiological processes. In this review, we focus on established and recent breakthroughs aimed at elucidating the impact of autophagy in differentiation and homeostasis maintenance of endothelium, muscle, immune system, and brain providing a suitable framework of the emerging results and highlighting the pivotal role of autophagic response in tissue functions, stem cell dynamics and differentiation rates.

Drp1 overexpression induces desmin disassembling and drives kinesin-1 activation promoting mitochondrial trafficking in skeletal muscle.

Mitochondria change distribution across cells following a variety of pathophysiological stimuli. The mechanisms presiding over this redistribution are yet undefined. In a murine model overexpressing Drp1 specifically in skeletal muscle, we find marked mitochondria repositioning in muscle fibres and we demonstrate that Drp1 is involved in this process. Drp1 binds KLC1 and enhances microtubule-dependent transport of mitochondria. Drp1-KLC1 coupling triggers the displacement of KIF5B from kinesin-1 complex increasing its binding to microtubule tracks and mitochondrial transport. High levels of Drp1 exacerbate this mechanism leading to the repositioning of mitochondria closer to nuclei. The reduction of Drp1 levels decreases kinesin-1 activation and induces the partial recovery of mitochondrial distribution. Drp1 overexpression is also associated with higher cyclin-dependent kinase-1 (Cdk-1) activation that promotes the persistent phosphorylation of desmin at Ser-31 and its disassembling. Fission inhibition has a positive effect on desmin Ser-31 phosphorylation, regardless of Cdk-1 activation, suggesting that induction of both fission and Cdk-1 are required for desmin collapse. This altered desmin architecture impairs mechanotransduction and compromises mitochondrial network stability priming mitochondria transport through microtubule-dependent trafficking with a mechanism that involves the Drp1-dependent regulation of kinesin-1 complex.

Mitophagy contributes to endothelial adaptation to simulated microgravity.

Exposure to real or simulated microgravity is sensed as a stress by mammalian cells, which activate a complex adaptive response. In human primary endothelial cells, we have recently shown the sequential intervention of various stress proteins which are crucial to prevent apoptosis and maintain cell function. We here demonstrate that mitophagy contributes to endothelial adaptation to gravitational unloading. After 4 and 10 d of exposure to simulated microgravity in the rotating wall vessel, the amount of BCL2 interacting protein 3, a marker of mitophagy, is increased and, in parallel, mitochondrial content, oxygen consumption, and maximal respiratory capacity are reduced, suggesting the acquisition of a thrifty phenotype to meet the novel metabolic challenges generated by gravitational unloading. Moreover, we suggest that microgravity induced-disorganization of the actin cytoskeleton triggers mitophagy, thus creating a connection between cytoskeletal dynamics and mitochondrial content upon gravitational unloading.

XIAP as a Target of New Small Organic Natural Molecules Inducing Human Cancer Cell Death.

X-linked inhibitor of apoptosis protein (XIAP) is an emerging crucial therapeutic target in cancer. We report on the discovery and characterisation of small organic molecules from Piper genus plants exhibiting XIAP antagonism, namely erioquinol, a quinol substituted in the 4-position with an alkenyl group and the alkenylphenols eriopodols A-C. Another isolated compound was originally identified as gibbilimbol B. Erioquinol was the most potent inhibitor of human cancer cell viability when compared with gibbilimbol B and eriopodol A was listed as intermediate. Gibbilimbol B and eriopodol A induced apoptosis through mitochondrial permeabilisation and caspase activation while erioquinol acted on cell fate via caspase-independent/non-apoptotic mechanisms, likely involving mitochondrial dysfunctions and aberrant generation of reactive oxygen species. In silico modelling and molecular approaches suggested that all molecules inhibit XIAP by binding to XIAP-baculoviral IAP repeat domain. This demonstrates a novel aspect of XIAP as a key determinant of tumour control, at the molecular crossroad of caspase-dependent/independent cell death pathway and indicates molecular aspects to develop tumour-effective XIAP antagonists.

3D Quantitative and Ultrastructural Analysis of Mitochondria in a Model of Doxorubicin Sensitive and Resistant Human Colon Carcinoma Cells.

Drug resistance remains a major obstacle in cancer treatment. Because mitochondria mediate metabolic reprogramming in cancer drug resistance, we focused on these organelles in doxorubicin sensitive and resistant colon carcinoma cells. We employed soft X-ray cryo nano-tomography to map three-dimensionally these cells at nanometer-resolution and investigate the correlation between mitochondrial morphology and drug resistance phenotype. We have identified significant structural differences in the morphology of mitochondria in the two strains of cancer cells, as well as lower amounts of Reactive oxygen species (ROS) in resistant than in sensitive cells. We speculate that these features could elicit an impaired mitochondrial communication in resistant cells, thus preventing the formation of the interconnected mitochondrial network as clearly detected in the sensitive cells. In fact, the qualitative and quantitative three-dimensional assessment of the mitochondrial morphology highlights a different structural organization in resistant cells, which reflects a metabolic cellular adaptation functional to survive to the offense exerted by the antineoplastic treatment.