Age-associated alterations of brain mitochondria energetics.

The study aimed to explore the role of age-associated elevated cytosolic Ca2+ in changes of brain mitochondria energetic processes. Two groups of rats, young adults (4 months) and advanced old (24 months), were evaluated for potential alterations of mitochondrial parameters, the oxidative phosphorylation (OxPhos), membrane potential, calcium retention capacity, activity of glutamate/aspartate carrier (aralar), and ROS formation. We demonstrated that the brain mitochondria of older animals have a lower resistance to Ca2+ stress with resulting consequences. The suppressed complex I OxPhos and decreased membrane potential were accompanied by reduction of the Ca2+ threshold required for induction of mPTP. The Ca2+ binding sites of mitochondrial aralar mediated a lower activity of old brain mitochondria. The altered interaction between aralar and mPTP may underlie mitochondrial dysregulation leading to energetic depression during aging. At the advanced stages of aging, the declined metabolism is accompanied by the diminished oxidative background.

Curcumin and Carnosic Acid Cooperate to Inhibit Proliferation and Alter Mitochondrial Function of Metastatic Prostate Cancer Cells.

Anticancer activities of plant polyphenols have been demonstrated in various models of neoplasia. However, evidence obtained in numerous in vitro studies indicates that proliferation arrest and/or killing of cancer cells require quite high micromolar concentrations of polyphenols that are difficult to reach in vivo and can also be (geno)toxic to at least some types of normal cells. The ability of certain polyphenols to synergize with one another at low concentrations can be used as a promising strategy to effectively treat human malignancies. We have recently reported that curcumin and carnosic acid applied at non-cytotoxic concentrations synergistically cooperate to induce massive apoptosis in acute myeloid leukemia cells, but not in normal hematopoietic and non-hematopoietic cells, via sustained cytosolic calcium overload. Here, we show that the two polyphenols can also synergistically suppress the growth of DU145 and PC-3 metastatic prostate cancer cell cultures. However, instead of cell killing, the combined treatment induced a marked inhibition of cell proliferation associated with G0/G1 cell cycle arrest. This was preceded by transient elevation of cytosolic calcium levels and prolonged dissipation of the mitochondrial membrane potential, without generating oxidative stress, and was associated with defective oxidative phosphorylation encompassing mitochondrial dysfunction. The above effects were concomitant with a significant downregulation of mRNA and protein expression of the oncogenic kinase SGK1, the mitochondria-hosted mTOR component. In addition, a moderate decrease in SGK1 phosphorylation at Ser422 was observed in polyphenol-treated cells. The mTOR inhibitor rapamycin produced a similar reduction in SGK1 mRNA and protein levels as well as phosphorylation. Collectively, our findings suggest that the combination of curcumin and carnosic acid at potentially bioavailable concentrations may effectively target different types of cancer cells by distinct modes of action. This and similar combinations merit further exploration as an anticancer modality.

Mitochondria membrane transformations in colon and prostate cancer and their biological implications.

Mitochondria have emerged as important determinants in cancer progression and malignancy. However, the role of mitochondrial membranes in cancer onset and progression has not been thoroughly investigated. This study compares the structural and functional properties of mitochondrial membranes in prostate and colon cancer cells in comparison to normal mitochondria, and possible therapeutic implications of these membrane changes. Specifically, isolation of cell mitochondria and preparation of inverted sub-mitochondrial particles (SMPs) illuminated significant cancer-induced modulations of membrane lipid compositions, fluidity, and activity of cytochrome c oxidase, one of the key mitochondrial enzymes. The experimental data further show that cancer-associated membrane transformations may account for mitochondria targeting by betulinic acid and resveratrol, known anti-cancer molecules. Overall, this study probes the relationship between cancer and mitochondrial membrane transformations, underlying a potential therapeutic significance for mitochondrial membrane targeting in cancer.

Mitochondrial responses to organelle-specific drug delivering nanoparticles composed of polypeptide and peptide complexes.

Aims: The mechanistic study of the drug carrier-target interactions of mitochondria-unique nanoparticles composed of polypeptide-peptide complexes (mPoP-NPs). Materials & methods: The isolated organelles were employed to address the direct effects of mPoP-NPs on dynamic structure and functional wellbeing of mitochondria. Mitochondria morphology, respiration, membrane potential, reactive oxygen species generation, were examined by confocal microscopy, flow cytometry and oxygraphy. Lonidamine-encapsulated formulation was assessed to evaluate the drug delivery capacity of the naive nanoparticles. Results: The mPoP-NPs do not alter mitochondria structure and performance upon docking to organelles, while successfully delivering drug that causes organelle dysfunction. Conclusion: The study gives insight into interactions of mPoP-NPs with mitochondria and provides substantial support for consideration of designed nanoparticles as biocompatible and efficient mitochondria-targeted platforms.

Malate-aspartate shuttle promotes l-lactate oxidation in mitochondria.

Metabolism in cancer cells is rewired to generate sufficient energy equivalents and anabolic precursors to support high proliferative activity. Within the context of these competing drives aerobic glycolysis is inefficient for the cancer cellular energy economy. Therefore, many cancer types, including colon cancer, reprogram mitochondria-dependent processes to fulfill their elevated energy demands. Elevated glycolysis underlying the Warburg effect is an established signature of cancer metabolism. However, there are a growing number of studies that show that mitochondria remain highly oxidative under glycolytic conditions. We hypothesized that activities of glycolysis and oxidative phosphorylation are coordinated to maintain redox compartmentalization. We investigated the role of mitochondria-associated malate-aspartate and lactate shuttles in colon cancer cells as potential regulators that couple aerobic glycolysis and oxidative phosphorylation. We demonstrated that the malate-aspartate shuttle exerts control over NAD+ /NADH homeostasis to maintain activity of mitochondrial lactate dehydrogenase and to enable aerobic oxidation of glycolytic l-lactate in mitochondria. The elevated glycolysis in cancer cells is proposed to be one of the mechanisms acquired to accelerate oxidative phosphorylation.

Cardiolipin mediates curcumin interactions with mitochondrial membranes.

Curcumin, the main molecular ingredient of the turmeric spice, has been reported to exhibit therapeutic properties for varied diseases and pathological conditions. While curcumin appears to trigger multiple signaling pathways, the precise mechanisms accounting for its therapeutic activity have not been deciphered. Here we show that curcumin exhibits significant interactions with cardiolipin (CL), a lipid exclusively residing in the mitochondrial membrane. Specifically, we found that curcumin affected the structures and dynamics of CL-containing biomimetic and biological mitochondrial membranes. Application of several biophysical techniques reveals the CL-promoted association and internalization of curcumin into lipid bilayers. In parallel, curcumin association with CL containing bilayers increased their fluidity and reduced lipid ordering. These findings suggest that membrane modifications mediated by CL interactions may play a role in the therapeutic functions of curcumin, and that the inner mitochondrial membrane in general might constitute a potential drug target.

Respirometric reserve capacity of cumulus cell mitochondria correlates with oocyte maturity.

PURPOSE: Oocyte competence is critical in success of assisted reproduction. Metabolic signaling between oocyte and cumulus cells within the cumulus-oocyte complex procure oocyte development. This study evaluated the relationship between respirometric activity of cumulus cells and maturity of corresponding oocytes. METHODS: In prospective cohort study, 20 women of age 28-42 undergoing IVF procedure were involved. To evaluate oocyte maturity, the cumulus cells from individual oocytes were assessed flow cytometrically by double labeling of cells with mitochondria specific dyes. The respirometric stress analysis using ATPase inhibitor oligomycin was applied to assess mitochondria metabolic abnormalities. RESULTS: The cumulus cells from each of 327 oocytes were analyzed. The respirometric index of cumulus cells (O'R) strongly correlates with maternal ovarian reserve, showing to be higher in patients with higher AMH (p < 0.0017). Cumulus cells from immature oocytes had severe mitochondria deficiency, i.e., low O'R, than those from mature oocytes (p < 0.02). No significant difference in respirometric capacity was found between cumulus cells associated with good vs poor-quality embryos. CONCLUSIONS: The oocyte maturity is potentially related to the mitochondria activity of cumulus cells.

Rapamycin increases oxidative metabolism and enhances metabolic flexibility in human cardiac fibroblasts.

Inhibition of mTOR signaling using rapamycin has been shown to increase lifespan and healthspan in multiple model organisms; however, the precise mechanisms for the beneficial effects of rapamycin remain uncertain. We have previously reported that rapamycin delays senescence in human cells and that enhanced mitochondrial biogenesis and protection from mitochondrial stress is one component of the benefit provided by rapamycin treatment. Here, using two models of senescence, replicative senescence and senescence induced by the presence of the Hutchinson-Gilford progeria lamin A mutation, we report that senescence is accompanied by elevated glycolysis and increased oxidative phosphorylation, which are both reduced by rapamycin. Measurements of mitochondrial function indicate that direct mitochondria targets of rapamycin are succinate dehydrogenase and matrix alanine aminotransferase. Elevated activity of these enzymes could be part of complex mechanisms that enable mitochondria to resume their optimal oxidative phosphorylation and resist senescence. This interpretation is supported by the fact that rapamycin-treated cultures do not undergo a premature senescence in response to the replacement of glucose with galactose in the culture medium, which forces a greater reliance on oxidative phosphorylation. Additionally, long-term treatment with rapamycin increases expression of the mitochondrial carrier protein UCP2, which facilitates the movement of metabolic intermediates across the mitochondrial membrane. The results suggest that rapamycin impacts mitochondrial function both through direct interaction with the mitochondria and through altered gene expression of mitochondrial carrier proteins.

Artefactual formation of pyruvate from in-source conversion of lactate.

RATIONALE: Lactate and pyruvate are high abundance products of glucose metabolism. Analysis of both molecules as part of metabolomics studies in cellular metabolism and physiology have been aided by advances in liquid chromatography-mass spectrometry (LC-MS). METHODS: We used ion pairing-chromatography and negative ion mode ESI on an QExactive HF to perform stable isotope assisted metabolomics profiling of lactate and pyruvate metabolism. RESULTS: Using an LC-MS method for polar metabolite analysis we discovered an artefactual formation of pyruvate from in-source fragmentation of lactate. Surprisingly, this in-source fragmentation has not been previously described, thus we report this identification to warn other investigators. This artefact was detected by baseline chromatographic resolution of lactate and pyruvate by LC with confirmation of this artefact by stable isotope labeling of lactate and pyruvate. CONCLUSIONS: These findings have immediate implications for metabolomics studies by LC-MS and direct infusion MS, especially in negative ion mode, whereby users should resolve lactate from pyruvate or robustly quantify the potential formation of pyruvate from higher abundance lactate in their assays.

Perfusion double-channel micropipette probes for oxygen flux mapping with single-cell resolution.

Measuring cellular respiration with single-cell spatial resolution is a significant challenge, even with modern tools and techniques. Here, a double-channel micropipette is proposed and investigated as a probe to achieve this goal by sampling fluid near the point of interest. A finite element model (FEM) of this perfusion probe is validated by comparing simulation results with experimental results of hydrodynamically confined fluorescent molecule diffusion. The FEM is then used to investigate the dependence of the oxygen concentration variation and the measurement signal on system parameters, including the pipette's shape, perfusion velocity, position of the oxygen sensors within the pipette, and proximity of the pipette to the substrate. The work demonstrates that the use of perfusion double-barrel micropipette probes enables the detection of oxygen consumption signals with micrometer spatial resolution, while amplifying the signal, as compared to sensors without the perfusion system. In certain flow velocity ranges (depending on pipette geometry and configuration), the perfusion flow increases oxygen concentration gradients formed due to cellular oxygen consumption. An optimal perfusion velocity for respiratory measurements on single cells can be determined for different system parameters (e.g., proximity of the pipette to the substrate). The optimum perfusion velocities calculated in this paper range from 1.9 to 12.5 µm/s. Finally, the FEM model is used to show that the spatial resolution of the probe may be varied by adjusting the pipette tip diameter, which may allow oxygen consumption mapping of cells within tissue, as well as individual cells at subcellular resolution.

Determination of mitochondrial metabolic phenotype through investigation of the intrinsic inhibition of succinate dehydrogenase.

Many diseases are accompanied by systemic or organ metabolic abnormalities. Therefore, investigation of the roles of mitochondrial dysfunction in the pathogenesis of major diseases requires a methodology that reflects the characteristics of mitochondrial metabolism particular for the organ of origin. We provide evidence that for brain and heart mitochondria the intrinsic inhibition of succinate dehydrogenase (SDH) is a key mechanism for attenuation of mitochondrial respiration and energy production in response to the organ's energy needs. This mechanism also serves to minimize the production of reactive oxygen species when the organ is at rest. Changes in the organ's workloads are accompanied by changes in metabolites that are used by mitochondria as substrates and for modification of energy production at the SDH level. Measurement of the respiratory activity of mitochondria with various substrates and substrate mixtures and use of bovine serum albumin as an SDH inhibitor will be useful for evaluation of metabolic phenotype at the mitochondrial level.

Danazol alters mitochondria metabolism of fibrocystic breast Mcf10A cells.

Fibrocystic Breast Disease (FBD) or Fibrocystic change (FC) affects about 60% of women at some time during their life. Although usually benign, it is often associated with pain and tenderness (mastalgia). The synthetic steroid danazol has been shown to be effective in reducing the pain associated with FBD, but the cellular and molecular mechanisms for its action have not been elucidated. We investigated the hypothesis that danazol acts by affecting energy metabolism. Effects of danazol on Mcf10A cells homeostasis, including mechanisms of oxidative phosphorylation, cytosolic calcium signaling and oxidative stress, were assessed by high-resolution respirometry and flow cytometry. In addition to fast physiological responses the associated genomic modulations were evaluated by Affimetrix microarray analysis. The alterations of mitochondria membrane potential and respiratory activity, downregulation of energy metabolism transcripts result in suppression of energy homeostasis and arrest of Mcf10A cells growth. The data obtained in this study impacts the recognition of direct control of mitochondria by cellular mechanisms associated with altered energy metabolism genes governing the breast tissue susceptibility and response to medication by danazol.

Mitochondria-Mediated Anticancer Effects of Non-Thermal Atmospheric Plasma.

Non-thermal atmospheric pressure plasma has attracted great interest due to its multiple potential biomedical applications with cancer treatment being among the most urgent. To realize the clinical potential of non-thermal plasma, the exact cellular and molecular mechanisms of plasma effects must be understood. This work aimed at studying the prostate cancer specific mechanisms of non-thermal plasma effects on energy metabolism as a central regulator of cell homeostasis and proliferation. It was found that cancer cells with higher metabolic rate initially are more resistant to plasma treated phosphate-buffered saline (PBS) since the respiratory and calcium sensitive signaling systems were not responsive to plasma exposure. However, dramatic decline of cancer oxidative phosphorylation developed over time resulted in significant progression of cell lethality. The normal prostate cells with low metabolic activity immediately responded to plasma treated PBS by suppression of respiratory functions and sustained elevation of cytosolic calcium. However, over time the normal cells start recovering their mitochondria functions, proliferate and restore the cell population. We found that the non-thermal plasma induced increase in intracellular ROS is of primarily non-mitochondrial origin. The discriminate non-thermal plasma effects hold a promise for clinical cancer intervention.

Tumor microenvironment promotes dicarboxylic acid carrier-mediated transport of succinate to fuel prostate cancer mitochondria.

Prostate cancer cells reprogram their metabolism, so that they support their elevated oxidative phosphorylation and promote a cancer friendly microenvironment. This work aimed to explore the mechanisms that cancer cells employ for fueling themselves with energy rich metabolites available in interstitial fluids. The mitochondria oxidative phosphorylation in metastatic prostate cancer DU145 cells and normal prostate epithelial PrEC cells were studied by high-resolution respirometry. An important finding was that prostate cancer cells at acidic pH 6.8 are capable of consuming exogenous succinate, while physiological pH 7.4 was not favorable for this process. Using specific inhibitors, it was demonstrated that succinate is transported in cancer cells by the mechanism of plasma membrane Na(+)-dependent dycarboxylic acid transporter NaDC3 (SLC13A3 gene). Although the level of expression of SLC13A3 was not significantly altered when maintaining cells in the medium with lower pH, the respirometric activity of cells under acidic condition was elevated in the presence of succinate. In contrast, normal prostate cells while expressing NaDC3 mRNA do not produce NaDC3 protein. The mechanism of succinate influx via NaDC3 in metastatic prostate cancer cells could yield a novel target for anti-cancer therapy and has the potential to be used for imaging-based diagnostics to detect non-glycolytic tumors.

Metabolic and structural integrity of magnetic nanoparticle-loaded primary endothelial cells for targeted cell therapy.

AIM: To successfully translate magnetically mediated cell targeting from bench to bedside, there is a need to systematically assess the potential adverse effects of magnetic nanoparticles (MNPs) interacting with 'therapeutic' cells. Here, we examined in detail the effects of internalized polymeric MNPs on primary rat endothelial cells' structural intactness, metabolic integrity and proliferation potential. MATERIALS & METHODS: The intactness of cytoskeleton and organelles was studied by fluorescent confocal microscopy, flow cytometry and high-resolution respirometry. RESULTS: MNP-loaded primary endothelial cells preserve intact cytoskeleton and organelles, maintain normal rate of proliferation, calcium signaling and mitochondria energy metabolism. CONCLUSION: This study provides supportive evidence that MNPs at doses necessary for targeting did not induce significant adverse effects on structural integrity and functionality of primary endothelial cells - potential cell therapy vectors.

The anti-cancer peptide, PNC-27, induces tumor cell necrosis of a poorly differentiated non-solid tissue human leukemia cell line that depends on expression of HDM-2 in the plasma membrane of these cells.

GOALS: We have developed the anti-cancer peptide, PNC-27, which is a membrane-active peptide that binds to the HDM-2 protein expressed in the cancer cell membranes of solid tissue tumor cells and induces transmembrane pore formation in cancer, but not in normal cells, resulting in tumor cell necrosis that is independent of p53 activity in these cells. We now extend our study to non-solid tissue tumor cells, in this case, a primitive, possible stem cell human leukemia cell line (K562) that is also p53-homozygously deleted. Our purpose was twofold: to investigate if these cells likewise express HDM-2 in their plasma membranes and to determine if our anti-cancer peptide induces tumor cell necrosis in these non-solid tissue tumor cells in a manner that depends on the interaction between the peptide and membrane-bound HDM-2. PROCEDURES: The anti-cancer activity and mechanism of PNC-27, which carries a p53 aa12-26-leader sequence connected on its carboxyl terminal end to a trans-membrane-penetrating sequence or membrane residency peptide (MRP), was studied against p53-null K562 leukemia cells. Murine leukocytes were used as a non-cancer cell control. Necrosis was determined by measuring the lactate dehydrogenase (LDH) release and apoptosis was determined by the detection of Caspases 3 and 7. Membrane colocalization of PNC-27 with HDM-2 was analyzed microscopically using fluorescently labeled antibodies against HDM-2 and PNC-27 peptides. RESULTS: We found that K562 cells strongly express HDM-2 protein in their membranes and that PNC-27 co-localizes with this protein in the membranes of these cells. PNC-27, but not the negative control peptide PNC-29, is selectively cytotoxic to K562 cells, inducing nearly 100 percent cell killing with LDH release. In contrast, this peptide had no effect on the lymphocyte control cells. CONCLUSIONS: The results suggest that HDM-2 is expressed in the membranes of non-solid tissue tumor cells in addition to the membranes of solid tissue tumor cells. Since K-562 cells appear to be in the stem cell family, the results suggest that early developing tumor cells also express HDM-2 protein in their membranes. Since PNC-27 induces necrosis of K-562 leukemia cells and co-localizes with HDM-2 in the tumor cell membrane as an early event, we conclude that the association of PNC-27 with HDM-2 in the cancer cell membrane results in trans-membrane pore formation which results in cancer cell death, as previously discovered in a number of different solid tissue tumor cells. Since K562 cells lack p53 expression, these effects of PNC-27 on this leukemia cell line occur by a p53-independent pathway.

Fatty acids in energy metabolism of the central nervous system.

In this review, we analyze the current hypotheses regarding energy metabolism in the neurons and astroglia. Recently, it was shown that up to 20% of the total brain's energy is provided by mitochondrial oxidation of fatty acids. However, the existing hypotheses consider glucose, or its derivative lactate, as the only main energy substrate for the brain. Astroglia metabolically supports the neurons by providing lactate as a substrate for neuronal mitochondria. In addition, a significant amount of neuromediators, glutamate and GABA, is transported into neurons and also serves as substrates for mitochondria. Thus, neuronal mitochondria may simultaneously oxidize several substrates. Astrocytes have to replenish the pool of neuromediators by synthesis de novo, which requires large amounts of energy. In this review, we made an attempt to reconcile ß-oxidation of fatty acids by astrocytic mitochondria with the existing hypothesis on regulation of aerobic glycolysis. We suggest that, under condition of neuronal excitation, both metabolic pathways may exist simultaneously. We provide experimental evidence that isolated neuronal mitochondria may oxidize palmitoyl carnitine in the presence of other mitochondrial substrates. We also suggest that variations in the brain mitochondrial metabolic phenotype may be associated with different mtDNA haplogroups.

One-dimensional nanoprobes for single-cell studies.

Owing to variation of individual cells within a population, single-cell studies are of great interest to researchers. Recent developments in nanofabrication technology have made this area increasingly attractive as one-dimensional (1D) nanoscale probes can be manufactured with increasing accuracy. Here, we provide an overview and description of the major designs that have been reported to date. For more details of what applications could be realized and how, based on the probe shapes and designs, we summarize the most recently reported performances of 1D single-cell probes with their advantages and limitations. Minimally invasive probes are required for long-term experiments on single cells. Carbon nanotubes with their unique properties and structure are excellent candidates for multitask robotic intracellular probes. Carbon nanotube-tipped cellular endoscopes are less invasive compared with pipettes or cantilever tips. Advances in nanofabrication techniques have made it possible to produce more consistent nanoscale cellular probes that can capture a variety of information from optical, electrical and chemical signals. In addition, these tools can transfer tiny amounts of fluids and molecular materials in a highly localized fashion for the purpose of analyzing or stimulating a variety of responses at the level of individual cells and even cellular organelles. We conclude with a critical analysis of the current state of the field as well as the major obstacles for further probe development of minimally invasive probes and their widespread use in cell biology.

Bioenergetic and antiapoptotic properties of mitochondria from cultured human prostate cancer cell lines PC-3, DU145 and LNCaP.

The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca²âº to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca²âº-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca²âº. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.