Mitochondria Deregulations in Cancer Offer Several Potential Targets of Therapeutic Interventions.

Mitochondria play a key role in cancer and their involvement is not limited to the production of ATP only. Mitochondria also produce reactive oxygen species and building blocks to sustain rapid cell proliferation; thus, the deregulation of mitochondrial function is associated with cancer disease development and progression. In cancer cells, a metabolic reprogramming takes place through a different modulation of the mitochondrial metabolic pathways, including oxidative phosphorylation, fatty acid oxidation, the Krebs cycle, glutamine and heme metabolism. Alterations of mitochondrial homeostasis, in particular, of mitochondrial biogenesis, mitophagy, dynamics, redox balance, and protein homeostasis, were also observed in cancer cells. The use of drugs acting on mitochondrial destabilization may represent a promising therapeutic approach in tumors in which mitochondrial respiration is the predominant energy source. In this review, we summarize the main mitochondrial features and metabolic pathways altered in cancer cells, moreover, we present the best known drugs that, by acting on mitochondrial homeostasis and metabolic pathways, may induce mitochondrial alterations and cancer cell death. In addition, new strategies that induce mitochondrial damage, such as photodynamic, photothermal and chemodynamic therapies, and the development of nanoformulations that specifically target drugs in mitochondria are also described. Thus, mitochondria-targeted drugs may open new frontiers to a tailored and personalized cancer therapy.

Mitofusin-2 Down-Regulation Predicts Progression of Non-Muscle Invasive Bladder Cancer.

Identification of markers predicting disease outcome is a major clinical issue for non-muscle invasive bladder cancer (NMIBC). The present study aimed to determine the role of the mitochondrial proteins Mitofusin-2 (Mfn2) and caseinolytic protease P (ClpP) in predicting the outcome of NMIBC. The study population consisted of patients scheduled for transurethral resection of bladder tumor upon the clinical diagnosis of bladder cancer (BC). Samples of the main bladder tumor and healthy-looking bladder wall from patients classified as NMIBC were tested for Mfn2 and ClpP. The expression levels of these proteins were correlated to disease recurrence, progression. Mfn2 and ClpP expression levels were significantly higher in lesional than in non-lesional tissue. Low-risk NMIBC had significantly higher Mfn2 expression levels and significantly lower ClpP expression levels than high-risk NMIBC; there were no differences in non-lesional levels of the two proteins. Lesional Mfn2 expression levels were significantly lower in patients who progressed whereas ClpP levels had no impact on any survival outcome. Multivariable analysis adjusting for the EORTC scores showed that Mfn2 downregulation was significantly associated with disease progression. In conclusion, Mfn2 and ClpP proteins were found to be overexpressed in BC as compared to non-lesional bladder tissue and Mfn2 expression predicted disease progression.

Mitochondrial Caseinolytic Protease P: A Possible Novel Prognostic Marker and Therapeutic Target in Cancer.

Caseinolytic protease P (ClpP) is a mitochondrial serine protease. In mammalian cells, the heterodimerization of ClpP and its AAA+ ClpX chaperone results in a complex called ClpXP, which has a relevant role in protein homeostasis and in maintaining mitochondrial functionality through the degradation of mitochondrial misfolded or damaged proteins. Recent studies demonstrate that ClpP is upregulated in primary and metastatic human tumors, supports tumor cell proliferation, and its overexpression desensitizes cells to cisplatin. Interestingly, small modulators of ClpP activity, both activators and inhibitors, are able to impair oxidative phosphorylation in cancer cells and to induce apoptosis. This review provides an overview of the role of ClpP in regulating mitochondrial functionality, in supporting tumor cell proliferation and cisplatin resistance; finally, we discuss whether this protease could represent a new prognostic marker and therapeutic target for the treatment of cancer.

Human Ovarian Cancer Tissue Exhibits Increase of Mitochondrial Biogenesis and Cristae Remodeling.

Ovarian cancer (OC) is the most lethal gynecologic cancer characterized by an elevated apoptosis resistance that, potentially, leads to chemo-resistance in the recurrent disease. Mitochondrial oxidative phosphorylation was found altered in OC, and mitochondria were proposed as a target for therapy. Molecular evidence suggests that the deregulation of mitochondrial biogenesis, morphology, dynamics, and apoptosis is involved in carcinogenesis. However, these mitochondrial processes remain to be investigated in OC. Eighteen controls and 16 OC tissues (serous and mucinous) were collected. Enzymatic activities were performed spectrophotometrically, mitochondrial DNA (mtDNA) content was measured by real-time-PCR, protein levels were determined by Western blotting, and mitochondrial number and structure were measured by electron microscopy. Statistical analysis was performed using Student's t-test, Mann-Whitney U test, and principal component analysis (PCA). We found, in OC, that increased mitochondrial number associated with increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and mitochondrial transcription factor A (TFAM) protein levels, as well as mtDNA content. The OC mitochondria presented an increased maximum length, as well as reduced cristae width and junction diameter, associated with increased optic atrophy 1 protein (OPA1) and prohibitin 2 (PHB2) protein levels. In addition, in OC tissues, augmented cAMP and sirtuin 3 (SIRT3) protein levels were observed. PCA of the 25 analyzed biochemical parameters classified OC patients in a distinct group from controls. We highlight a "mitochondrial signature" in OC that could result from cooperation of the cAMP pathway with the SIRT3, OPA1, and PHB2 proteins.

Mitochondrial Dysfunctions in Type I Endometrial Carcinoma: Exploring Their Role in Oncogenesis and Tumor Progression.

Type I endometrial cancer (EC) is the most common form of EC, displaying less aggressive behavior than type II. The development of type I endometrial cancer is considered a multistep process, with slow progression from normal endometrium to hyperplasia, the premalignant form, and endometrial cancer as a result of an unopposed estrogenic stimulation. The role of mitochondria in type I EC tumor progression and prognosis is currently emerging. This review aims to explore mitochondrial alterations in this cancer and in endometrial hyperplasia focusing on mitochondrial DNA mutations, respiratory complex I deficiency, and the activation of mitochondrial quality control systems. A deeper understanding of altered mitochondrial pathways in type I EC could provide novel opportunities to discover new diagnostic and prognostic markers as well as potential therapeutic targets.

Growth hormone secretagogues hexarelin and JMV2894 protect skeletal muscle from mitochondrial damages in a rat model of cisplatin-induced cachexia.

Chemotherapy can cause cachexia, which consists of weight loss associated with muscle atrophy. The exact mechanisms underlying this skeletal muscle toxicity are largely unknown and co-therapies to attenuate chemotherapy-induced side effects are lacking. By using a rat model of cisplatin-induced cachexia, we here characterized the mitochondrial homeostasis in tibialis anterior cachectic muscle and evaluated the potential beneficial effects of the growth hormone secretagogues (GHS) hexarelin and JMV2894 in this setting. We found that cisplatin treatment caused a decrease in mitochondrial biogenesis (PGC-1α, NRF-1, TFAM, mtDNA, ND1), mitochondrial mass (Porin and Citrate synthase activity) and fusion index (MFN2, Drp1), together with changes in the expression of autophagy-related genes (AKT/FoxO pathway, Atg1, Beclin1, LC3AII, p62) and enhanced ROS production (PRX III, MnSOD). Importantly, JMV2894 and hexarelin are capable to antagonize this chemotherapy-induced mitochondrial dysfunction. Thus, our findings reveal a key-role played by mitochondria in the mechanism responsible for GHS beneficial effects in skeletal muscle, strongly indicating that targeting mitochondrial dysfunction might be a promising area of research in developing therapeutic strategies to prevent or limit muscle wasting in cachexia.

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

Dietary supplementation with acetyl-l-carnitine counteracts age-related alterations of mitochondrial biogenesis, dynamics and antioxidant defenses in brain of old rats.

We previously reported the ability of dietary supplementation with acetyl-l-carnitine (ALCAR) to prevent age-related decreases of mitochondrial biogenesis in skeletal muscle and liver of old rats. Here, we investigate the effects of ALCAR supplementation in cerebral hemispheres and cerebellum of old rats by analyzing several parameters linked to mitochondrial biogenesis, mitochondrial dynamics and antioxidant defenses. We measured the level of the coactivators PGC-1α and PGC-1ß and of the factors regulating mitochondrial biogenesis, finding an age-related decrease of PGC-1ß, whereas PGC-1α level was unvaried. Twenty eight-month old rats supplemented with ALCAR for one and two months showed increased levels of both factors. Accordingly, the expression of the two transcription factors NRF-1 and TFAM followed the same trend of PGC-1ß. The level of mtDNA, ND1 and the activity of citrate synthase, were decreased with aging and increased following ALCAR treatment. Furthermore, ALCAR counteracted the age-related increase of deleted mtDNA. We also analyzed the content of proteins involved in mitochondrial dynamics (Drp1, Fis1, OPA1 and MNF2) and found an age-dependent increase of MFN2 and of the long form of OPA1. ALCAR treatment restored the content of the two proteins to the level of the young rats. No changes with aging and ALCAR were observed for Drp1 and Fis1. ALCAR reduced total cellular levels of oxidized PRXs and counteracted the age-related decrease of PRX3 and SOD2. Overall, our findings indicate a systemic positive effect of ALCAR dietary treatment and a tissue specific regulation of mitochondrial homeostasis in brain of old rats. Moreover, it appears that ALCAR acts as a nutrient since in most cases its effects were almost completely abolished one month after treatment suspension. Dietary supplementation of old rats with this compound seems a valuable approach to prevent age-related mitochondrial dysfunction and might ultimately represent a strategy to delay age-associated negative consequences in mitochondrial homeostasis.

Mitochondrial dysfunctions in bladder cancer: Exploring their role as disease markers and potential therapeutic targets.

Bladder cancer (BC) is a major cause of mortality worldwide as it currently lacks fully reliable markers of disease outcome and effective molecular targets for therapy. Mitochondria play a key role in cell metabolism but the role of mitochondrial dysfunctions in BC has been scarcely investigated. In this review, we explored current evidence for the potential role of mitochondrial DNA (mtDNA) alterations (point mutations and copy number) as disease markers in BC. Some germline mtDNA mutations detectable in blood could represent a non-invasive tool to predict the risk of developing BC. MtDNA copy number and tumor specific mtDNA mutations and RNAs showed encouraging results as novel molecular markers for early detection of BC in body fluids. Moreover, mitochondrial proteins Lon protease, Mitofusin-2, and TFAM may have prognostic/predictive value and may represent potential therapeutic targets. A deeper understanding of mitochondrial dysfunctions in BC could therefore provide novel opportunities for targeted therapeutic strategies.

Increase in proteins involved in mitochondrial fission, mitophagy, proteolysis and antioxidant response in type I endometrial cancer as an adaptive response to respiratory complex I deficiency.

Pathogenic mtDNA mutations associated with alterations of respiratory complex I, mitochondrial proliferation (oncocytic-like phenotype) and increase in antioxidant response were previously reported in type I endometrial carcinoma (EC). To evaluate whether in the presence of pathogenic mtDNA mutations other mitochondrial adaptive processes are triggered by cancer cells, the expression level of proteins involved in mitochondrial dynamics, mitophagy, proteolysis and apoptosis were evaluated in type I ECs harboring pathogenic mtDNA mutations and complex I deficiency. An increase in the fission protein Drp1, in the mitophagy protein BNIP3, in the mitochondrial protease CLPP, in the antioxidant and anti-apoptotic protein ALR and in Bcl-2 as well as a decrease in the fusion protein Mfn2 were found in cancer compared to matched non malignant tissue. Moreover, the level of these proteins was measured in type I EC, in hyperplastic (the premalignant form) and in non malignant tissues to verify whether the altered expression of these proteins is a common feature of endometrial cancer and of hyperplastic tissue. This analysis confirmed in type I EC samples, but not in hyperplasia, an alteration of the expression level of these proteins. These results suggest that in this cancer mitochondrial fission, antioxidant and anti-apoptotic response may be activated, as well as the discharge of damaged mitochondrial proteins as adaptation processes to mitochondrial dysfunction.

Inhibition of Lon protease by triterpenoids alters mitochondria and is associated to cell death in human cancer cells.

Mitochondrial Lon protease (Lon) regulates several mitochondrial functions, and is inhibited by the anticancer molecule triterpenoid 2-cyano-3, 12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), or by its C-28 methyl ester derivative (CDDO-Me). To analyze the mechanism of action of triterpenoids, we investigated intramitochondrial reactive oxygen species (ROS), mitochondrial membrane potential, mitochondrial mass, mitochondrial dynamics and morphology, and Lon proteolytic activity in RKO human colon cancer cells, in HepG2 hepatocarcinoma cells and in MCF7 breast carcinoma cells. We found that CDDO and CDDO-Me are potent stressors for mitochondria in cancer cells, rather than normal non-transformed cells. In particular, they: i) cause depolarization; ii) increase mitochondrial ROS, iii) alter mitochondrial morphology and proteins involved in mitochondrial dynamics; iv) affect the levels of Lon and those of aconitase and human transcription factor A, which are targets of Lon activity; v) increase level of protein carbonyls in mitochondria; vi) lead to intrinsic apoptosis. The overexpression of Lon can rescue cells from cell death, providing an additional evidence on the role of Lon in conditions of excessive stress load.

Mitochondrial changes in endometrial carcinoma: possible role in tumor diagnosis and prognosis (review).

Endometrial carcinoma (EC) is a solid neoplasia for which a role for mitochondria in cancer progression is currently emerging and yet represents a diagnostic and prognostic challenge. EC is one of the most frequently occurring gynecological malignancies in the Western world whose incidence has increased significantly during the last decades. Here, we review the literature data on mitochondrial changes reported in EC, namely, mitochondrial DNA (mtDNA) mutations, increase in mitochondrial biogenesis and discuss whether they may be used as new cancer biomarkers for early detection and prognosis of this cancer.

Analysis of the mitochondrial proteome of cybrid cells harbouring a truncative mitochondrial DNA mutation in respiratory complex I.

Transmitochondrial cytoplasmic hybrids (cybrids) are well established model systems to reveal the effects of mitochondrial DNA (mtDNA) mutations on cell metabolism excluding the interferences of a different nuclear background. The m.3571insC mutation in the MTND1 gene of respiratory complex I (CI) is commonly detected in oncocytic tumors, in which it causes a severe CI dysfunction leading to an energetic impairment when present above 83% mutant load. To assess whether the energetic deficit may alter the mitochondrial proteome, OS-78 and OS-93 cybrid cell lines bearing two different degrees of the m.3571insC mutation (78% and 92.8%, respectively) and control cybrids bearing wild-type mtDNA (CC) were analyzed. Two-dimensional electrophoresis and mass spectrometry revealed significant alterations only in cybrids above the threshold (OS-93). All differentially expressed proteins are decreased. In particular, the levels of the pyruvate dehydrogenase E1 chain B subunit (E1ß), of lipoamide dehydrogenase (E3), the enzyme component of pyruvate and 2-oxoglutarate dehydrogenase complexes, and of lactate dehydrogenase B (LDHB) were reduced. Moreover, a significant decrease of the pyruvate dehydrogenase complex activity was found when OS-93 cybrid cells were grown in galactose medium, a metabolic condition that forces cells to use respiration. These results demonstrate that the energetic impairment caused by the almost homoplasmic m.3571insC mutation perturbs cellular metabolism leading to a decreased steady state level of components of very important mitochondrial NAD-dependent dehydrogenases.

Acetyl-L-carnitine activates the peroxisome proliferator-activated receptor-γ coactivators PGC-1α/PGC-1ß-dependent signaling cascade of mitochondrial biogenesis and decreases the oxidized peroxiredoxins content in old rat liver.

The behavior of the peroxisome proliferator-activated receptor-γ coactivators PGC-1α/PGC-ß-dependent mitochondrial biogenesis signaling pathway, as well as the level of some antioxidant enzymes and proteins involved in mitochondrial dynamics in the liver of old rats before and after 2 months of acetyl-L-carnitine (ALCAR) supplementation, was tested. The results reveal that ALCAR treatment is able to reverse the age-associated decline of PGC-1α, PGC-1ß, nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 (ND1), and cytochrome c oxidase subunit IV (COX IV) protein levels, of mitochondrial DNA (mtDNA) content, and of citrate synthase activity. Moreover, it partially reverses the mitochondrial superoxide dismutase 2 (SOD2) decline and reduces the cellular content of oxidized peroxiredoxins. These data demonstrate that ALCAR treatment is able to promote in the old rat liver a new mitochondrial population that can contribute to the cellular oxidative stress reduction. Furthermore, a remarkable decline of Drp1 and of Mfn2 proteins is reported here for the first time, suggesting a reduced mitochondrial dynamics in aging liver with no effect of ALCAR treatment.

Rat liver mitochondrial proteome: changes associated with aging and acetyl-L-carnitine treatment.

Oxidative stress has a central role in aging and in several age-linked diseases such as neurodegenerative diseases, diabetes and cancer. Mitochondria, as the main cellular source and target of reactive oxygen species (ROS) in aging, are recognized as very important players in the above reported diseases. Impaired mitochondrial oxidative phosphorylation has been reported in several aging tissues. Defective mitochondria are not only responsible of bioenergetically less efficient cells but also increase ROS production further contributing to tissues oxidative stress. Acetyl-L-carnitine (ALCAR) is a biomolecule able to limit age-linked mitochondrial decay in brain, liver, heart and skeletal muscles by increasing mitochondrial efficiency. Here the global changes induced by aging and by ALCAR supplementation to old rat on the mitochondrial proteome of rat liver has been analyzed by means of the two-dimensional polyacrylamide gel electrophoresis. Mass spectrometry has been used to identify the differentially expressed proteins. A significant age-related change occurred in 31 proteins involved in several metabolisms. ALCAR supplementation altered the levels of 26 proteins. In particular, ALCAR reversed the age-related alterations of 10 mitochondrial proteins relative to mitochondrial cristae morphology, to the oxidative phosphorylation and antioxidant systems, to urea cycle, to purine biosynthesis.

Accumulation of overoxidized Peroxiredoxin III in aged rat liver mitochondria.

Overoxidation and subsequent inactivation of Peroxiredoxin III (PrxIII), a mitochondrial H(2)O(2) scavenging enzyme, have been reported in oxidative stress conditions. No data are available in the literature about the presence of overoxidized forms of PrxIII in aged tissues. Liver mitochondria from 12-month-old rats and 28-month-old rats were here analyzed by two-dimensional gel electrophoresis. A spot corresponding to the native form of PrxIII was present in adult and old rats with the same volume, whereas an additional, more acidic spot, of the same molecular weight of the native form, accumulated only in old rats. The acidic spot was identified, by MALDI-MS analysis, as a form of PrxIII bearing the cysteine of the catalytic site overoxidized to sulphonic acid. This modified PrxIII form corresponds to the irreversibly inactivated enzyme, here reported, for the first time, in aging. Three groups of 28-month-old rats treated with acetyl-l-carnitine were also examined. Reduced accumulation of the overoxidized PrxIII form was found in all ALCAR-treated groups.

A DIGE approach for the assessment of rat soleus muscle changes during unloading: effect of acetyl-L-carnitine supplementation.

After hind limb suspension, a remodeling of postural muscle phenotype is observed. This remodeling results in a shift of muscle profile from slow-oxidative to fast-glycolytic. These metabolic changes and fiber type shift increase muscle fatigability. Acetyl-L-carnitine (ALCAR) influences the skeletal muscle phenotype of soleus muscle suggesting a positive role of dietary supplementation of ALCAR during unloading. In the present study, we applied a 2-D DIGE, mass spectrometry and biochemical assays, to assess qualitative and quantitative differences in the proteome of rat slow-twitch soleus muscle subjected to disuse. Meanwhile, the effects of ALCAR administration on muscle proteomic profile in both unloading and normal-loading conditions were evaluated. The results indicate a modulation of troponin I and tropomyosin complex to regulate fiber type transition. Associated, or induced, metabolic changes with an increment of glycolytic enzymes and a decreased capacity of fat oxidation are observed. These metabolic changes appear to be counteracted by ALCAR treatment, which restores the mitochondrial mass and decreases the glycolytic enzyme expression, suggesting a normalization of the metabolic shift observed in unloaded animals. This normalization is accompanied by a maintenance of body weight and seems to prevent a switch of fiber type.

DmTTF, a novel mitochondrial transcription termination factor that recognises two sequences of Drosophila melanogaster mitochondrial DNA.

Using a combination of bioinformatic and molecular biology approaches a Drosophila melanogaster protein, DmTTF, has been identified, which exhibits sequence and structural similarity with two mitochondrial transcription termination factors, mTERF (human) and mtDBP (sea urchin). Import/processing assays indicate that DmTTF is synthesised as a precursor of 410 amino acids and is imported into mitochondria, giving rise to a mature product of 366 residues. Band-shift and DNase I protection experiments show that DmTTF binds two homologous, short, non-coding sequences of Drosophila mitochondrial DNA, located at the 3' end of blocks of genes transcribed on opposite strands. The location of the target sequences coincides with that of two of the putative transcription termination sites previously hypothesised. These results indicate that DmTTF is the termination factor of mitochondrial transcription in Drosophila. The existence of two DmTTF binding sites might serve not only to stop transcription but also to control the overlapping of a large number of transcripts generated by the peculiar transcription mechanism operating in this organism.

Acetylation and level of mitochondrial transcription factor A in several organs of young and old rats.

To gain further information on the role of mitochondrial transcription factor A (TFAM) in mitochondrial biogenesis, we studied the post-translational modifications of the protein in 6- and 28-month-old rat liver. Mass spectrometry and immunoblot analysis revealed that TFAM was acetylated at a single lysine residue and that the level of acetylation did not change with age. The measurement of the content of TFAM and of mitochondrial DNA (mtDNA) in several organs (cerebellum, heart, kidney, and liver) of young and old rats showed an age-related increase of mtDNA and TFAM in all the organs analyzed, except in heart. These data are discussed in the light of the multiple roles of TFAM in mitochondrial biogenesis and of the age-related change of the mitochondrial transcription.