Lower Mitochondrial Membrane Potential Research Articles

Live Cell Imaging Reveals pH Oscillations in Saccharomyces cerevisiae During Metabolic Transitions

Addition of glucose to starved Saccharomyces cerevisiae initiates collective NADH dynamics termed glycolytic oscillations. Numerous questions remain about the extent to which single cells can oscillate, if oscillations occur in natural conditions, and potential physiological consequences of oscillations. In this paper, we report sustained glycolytic oscillations in single cells without the need for cyanide. Glucose addition to immobilized cells induced pH oscillations that could be imaged with fluorescent sensors. A population of cells had oscillations that were heterogeneous in frequency, start time, stop time, duration and amplitude. These changes in cytoplasmic pH were necessary and sufficient to drive changes in NADH. Oscillators had lower mitochondrial membrane potentials and budded more slowly than non-oscillators. We also uncovered a new type of oscillation during recovery from H2O2 challenge. Our data show that pH in S. cerevisiae changes over several time scales, and that imaging pH offers a new way to measure glycolytic oscillations on individual cells.

Characterization of subventricular zone-derived progenitor cells from mild and late symptomatic YAC128 mouse model of Huntington's disease

Huntington's disease (HD) is caused by an expansion of CAG repeats in the HTT gene, leading to expression of mutant huntingtin (mHTT) and selective striatal neuronal loss, frequently associated with mitochondrial dysfunction and decreased support of brain-derived neurotrophic factor (BDNF). New neurons derived from the subventricular zone (SVZ) are apparently not able to rescue HD pathological features. Thus, we analyzed proliferation, migration and differentiation of adult SVZ-derived neural stem/progenitor cells (NSPC) from mild (6month-old (mo)) and late (10mo) symptomatic HD YAC128 mice expressing full-length (FL)-mHTT versus age-matched wild-type (WT) mice. SVZ cells derived from 6mo YAC128 mice exhibited higher migratory capacity and a higher number of MAP2+ and synaptophysin+cells, compared to WT cells; MAP2 labeling was enhanced after exposure to BDNF. However, BDNF-evoked neuronal differentiation was not observed in 10mo YAC128 SVZ-derived cells. Interestingly, 6mo YAC128 SVZ-derived cells showed increased intracellular Ca2+ levels in response to KCl, which was potentiated by BDNF, evidencing the presence of differentiated neurons. In contrast, KCl depolarization-induced intracellular Ca2+ increase in 10mo YAC128 SVZ-derived cells was shown to be increased only in BDNF-treated YAC128 SVZ-derived cells, suggestive of decreased differentiation capacity. In addition, BDNF-untreated NSPC from 10mo YAC128 mice exhibited lower mitochondrial membrane potential and increased mitochondrial Ca2+ accumulation, in relation with NSPC from 6mo YAC128 mice. Data evidence age-dependent reduced migration and decreased acquisition of a neuronal phenotype, accompanied by decreased mitochondrial membrane potential in SVZ-derived cells from YAC128 mice through HD symptomatic phases.

High-intensity Interval Training Improves Mitochondrial Function and Suppresses Thrombin Generation in Platelets undergoing Hypoxic Stress

This study elucidates how high-intensity interval training (HIT) and moderate-intensity continuous training (MCT) affect mitochondrial functionality and thrombin generation (TG) in platelets following hypoxic exercise (HE, 100 W under 12% O2 for 30 min). Forty-five healthy sedentary males were randomized to engage either HIT (3-minute intervals at 40% and 80%VO2max, n = 15) or MCT (sustained 60%VO2max, n = 15) for 30 minutes/day, 5 days/week for 6 weeks, or to a control group (CTL, n = 15) that did not received exercise intervention. Before the intervention, HE (i) reduced the ATP-linked O2 consumption rate (OCR), the reserve capacity of OCR, and the activities of citrate synthase (CS) and succinate dehydrogenase (SDH), (ii) lowered mitochondrial membrane potential (MP) and elevated matrix oxidant burden (MOB) in platelets, and (iii) enhanced dynamic TG in platelet-rich plasma (PRP), which responses were attenuated by pretreating PRP with oligomycin or rotenone/antimycin A. However, 6-week HIT (i) increased mitochondrial OCR capacity with enhancing the CS and SDH activities and (ii) heightened mitochondrial MP with depressing MOB in platelets following HE, compared to those of MCT and CTL. Moreover, the HIT suppressed the HE-promoted dynamic TG in PRP. Hence, we conclude that the HIT simultaneously improves mitochondrial bioenergetics and suppresses dynamic TG in platelets undergoing hypoxia.

SLC4A11 depletion impairs NRF2 mediated antioxidant signaling and increases reactive oxygen species in human corneal endothelial cells during oxidative stress

Corneal endothelial dystrophy is a progressive disease with gradual loss of vision and characterized by degeneration and dysfunction of corneal endothelial cells. Mutations in SLC4A11, a Na+ dependent OH− transporter, cause congenital hereditary endothelial dystrophy (CHED) and Fuchs’ endothelial corneal dystrophy (FECD), the two most common forms of endothelial degeneration. Along with genetic factors, oxidative stress plays a role in pathogenesis of several corneal diseases. In this study we looked into the role of SLC4A11 in antioxidant stress response in human corneal endothelial cells (HCEnC). We found increased expression of SLC4A11 in presence of oxidative stress. Depletion of SLC4A11 using targeted siRNA, caused an increase in reactive oxygen species, cytochrome c, lowered mitochondrial membrane potential, and reduced cell viability during oxidative stress. Moreover, SLC4A11 was found to be necessary for NRF2 mediated antioxidant gene expression in HCEnC. On the other hand, over expression of SLC4A11 reduces reactive oxygen species levels and increases cell viability. Lastly, CHED tissue specimens show evidence of oxidative stress and reduced expression of NRF2. In conclusion, our data suggests a possible role of SLC4A11 in regulating oxidative stress, and might be responsible for both the etiology and treatment of corneal endothelial dystrophy.

Copper doping enhanced the oxidative stress-mediated cytotoxicity of TiO2 nanoparticles in A549 cells.

Physicochemical properties of titanium dioxide nanoparticles (TiO2 NPs) can be tuned by doping with metals or nonmetals. Copper (Cu) doping improved the photocatalytic behavior of TiO2 NPs that can be applied in various fields such as environmental remediation and nanomedicine. However, interaction of Cu-doped TiO2 NPs with human cells is scarce. This study was designed to explore the role of Cu doping in cytotoxic response of TiO2 NPs in human lung epithelial (A549) cells. Characterization data demonstrated the presence of both TiO2 and Cu in Cu-doped TiO2 NPs with high-quality lattice fringes without any distortion. The size of Cu-doped TiO2 NPs (24 nm) was lower than pure TiO2 NPs (30 nm). Biological results showed that both pure and Cu-doped TiO2 NPs induced cytotoxicity and oxidative stress in a dose-dependent manner. Low mitochondrial membrane potential and higher caspase-3 enzyme (apoptotic markers) activity were also observed in A549 cells exposed to pure and Cu-doped TiO2 NPs. We further observed that cytotoxicity caused by Cu-doped TiO2 NPs was higher than pure TiO2 NPs. Moreover, antioxidant N-acetyl cysteine effectively prevented the reactive oxygen species generation, glutathione depletion, and cell viability reduction caused by Cu-doped TiO2 NPs. This is the first report showing that Cu-doped TiO2 NPs induced cytotoxicity and oxidative stress in A549 cells. This study warranted further research to explore the role of Cu doping in toxicity mechanisms of TiO2 NPs.

α-Pyrrolidinononanophenone provokes apoptosis of neuronal cells through alterations in antioxidant properties.

In this study, we found that exposure to α-pyrrolidinononanophenone (α-PNP), a highly lipophilic synthetic cathinone, provokes apoptosis of human neuronal SK-N-SH cells. The drug sensitivity of the cells (50% lethal concentration of 12μM) was similar to those of aortic endothelial and smooth muscle cells, and was higher than those of cells derived from colon, liver, lung and kidney, suggesting that α-PNP overdose and abuse cause serious damage in central nervous and vascular systems. SK-N-SH cell treatment with lethal concentrations (20 and 50μM) of α-PNP facilitated the reactive oxygen species (ROS) production. The treatment also prompted elevation of Bax/Bcl-2 ratio, lowering of mitochondrial membrane potential, release of cytochrome-c into cytosol, and resultant activation of caspase-9 and caspase-3. The apoptotic events (caspase-3 activation and DNA fragmentation) were abolished by pretreatment with antioxidants, N-acetyl-l-cysteine and polyethyleneglycol-conjugated catalase. These results suggest that ROS production, mitochondrial dysfunction and caspase activation are potential events in the mechanism underlying the α-PNP-triggered neuronal cell apoptosis. Intriguingly, the α-PNP treatment of SK-N-SH cells was found to promote formation of 4-hydroxynonenal, a reactive aldehyde generated from lipid peroxidation. The α-PNP treatment also decreased cellular levels of total and reduced glutathiones, expression of γ-glutamylcysteine synthetase mRNA and glutathione reductase activity. Furthermore, the α-PNP treatment resulted in both decrease in proteasomal activities and increase in expression of autophagy-related factors, which were significantly prevented by pretreating with N-acetyl-l-cysteine. Therefore, the ROS formation by α-PNP treatment may be ascribable to the decrease in glutathione level through its consumption during 4-hydroxynonenal detoxification and dysfunction of both de novo synthesis and regeneration of glutathione, in addition to impairments in proteasomal and autophagic systems that degrade cellular oxidized components.

The Association between the C5263T Mutation in the Mitochondrial ND2 Gene and Coronary Heart Disease among Young Chinese Han People

ObjectiveThis study aimed to investigate the genetic background of mitochondrial genes in young patients with Coronary heart disease (CHD) to provide a foundation for the early prevention of young patients with CHD. Methods115 cases of young (≤ 45 years) CHD Chinese Han patients (case group), 100 cases of older (> 45 years) Chinese Han CHD patients (experimental group) hospitalized and 100 cases of healthy people through physical examination (control group) at the General Hospital of PLA between January 2014 and December 2015 were selected. General information, clinical assessment, pedigree analysis, and mitochondrial full sequence scanning were performed. The pedigrees of one patient harbouring the C5263T mutation were recruited. Mitochondrial functional analysis including cellular reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) were performed on pedigrees with the C5263T mutation (mutation group) and without the mutation (non-mutation group). ResultsThe differences in biochemical tests (P > 0.05) between the case group and experimental group were not significant. The C5263T single-nucleotide mutation of the mitochondrial ND2 gene was observed in 2 young CHD patients in the case group. The premature CHD of these 2 patients followed a pattern of maternal inheritance. The mutation group (I1, II2) had higher ROS levels (4750.82 ± 1045.55 vs. 3888.58 ± 487.60, P = 0.022) and lower MMP levels (P = 0.045) than the non-mutation group (II1, III1, III2). ConclusionWe speculated that the mitochondrial C5263T mutation might be associated with the occurrence CHD in Chinese Han young people.

Pathogenic p62/SQSTM1 mutations impair energy metabolism through limitation of mitochondrial substrates

Abnormal mitochondrial function has been found in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Mutations in the p62 gene (also known as SQSTM1) which encodes the p62 protein have been reported in both disorders supporting the idea of an ALS/FTD continuum. In this work the role of p62 in energy metabolism was studied in fibroblasts from FTD patients carrying two independent pathogenic mutations in the p62 gene, and in a p62-knock-down (p62 KD) human dopaminergic neuroblastoma cell line (SH-SY5Y). We found that p62 deficiency is associated with inhibited complex I mitochondrial respiration due to lack of NADH for the electron transport chain. This deficiency was also associated with increased levels of NADPH reflecting a higher activation of pentose phosphate pathway as this is accompanied with higher cytosolic reduced glutathione (GSH) levels. Complex I inhibition resulted in lower mitochondrial membrane potential and higher cytosolic ROS production. Pharmacological activation of transcription factor Nrf2 increased mitochondrial NADH levels and restored mitochondrial membrane potential in p62-deficient cells. Our results suggest that the phenotype is caused by a loss-of-function effect, because similar alterations were found both in the mutant fibroblasts and the p62 KD model. These findings highlight the implication of energy metabolism in pathophysiological events associated with p62 deficiency.

Effects of dihydroartemisinin on radiosensitivity of Raji cells

To study the effects of dihydroartemisinin (DHA) on radiation sensitivity of Raji cells, and explore its mechanisms. CCK8 was used to determine the effect of DHA on cell viability of Raji cells; apoptosis, intracellular reactive oxygen speies(ROS) and mitochondrial membrane potential of Raji cells were detected by flow cytometry; and the protein expressions of protein kinase B(AKT), phospho-rylated-protein kinase B(p-AKT), Bcl-2 and Bax were determined by Western blot. The cells were randomly divided into four groups:control group, DHA(5μmol/L DHA), irradiation(IR, 4 Gy), IR+DHA group (4 Gy IR+5 μmol/L DHA). Compared with the other three groups, cells in DHA+IR group exhibited lower mitochondrial membrane potential (P<0.01). While the intracellular ROS content and apoptosis rate of Raji cells in DHA+IR group were increased significantly(P<0.01). In addition, compared with the other three groups, there was no significant difference in the expression of AKT, but the phosphorylation of AKT protein were significantly inhibited and the expression of Bcl-2 protein was markedly decreased. However, the expressions of Bax and Cleaved-Caspase-3 protein were markedly increased. DHA might activate the mitochondrial apoptotic signal via inhibiting phosphoinositide 3-kinase (PI3K/AKT) pathway and increase oxidative stress to enhance the radiosensitivity of Raji cells.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies.

Obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy

Mitochondria are the most prominent organelle in the oocyte. Somatic cells maintain a healthy population of mitochondria by degrading damaged mitochondria via mitophagy, a specialized autophagy pathway. However, evidence from previous work investigating the more general macroautophagy pathway in oocytes suggests that mitophagy may not be active in the oocyte. This would leave the vast numbers of mitochondria – poised to be inherited by the offspring – vulnerable to damage. Here we test the hypothesis that inactive mitophagy in the oocyte underlies maternal transmission of dysfunctional mitochondria. To determine whether oocytes can complete mitophagy, we used either CCCP or AntimycinA to depolarize mitochondria and trigger mitophagy. After depolarization, we did not detect co-localization of mitochondria with autophagosomes and mitochondrial DNA copy number remained unchanged, indicating the non-functional mitochondrial population was not removed. To investigate the impact of an absence of mitophagy in oocytes with damaged mitochondria on offspring mitochondrial function, we utilized in vitro fertilization of high fat high sugar (HF/HS)-exposed oocytes, which have lower mitochondrial membrane potential and damaged mitochondria. Here, we demonstrate that blastocysts generated from HF/HS oocytes have decreased mitochondrial membrane potential, lower metabolites involved in ATP generation, and accumulation of PINK1, a mitophagy marker protein. This mitochondrial phenotype in the blastocyst mirrors the phenotype we show in HF/HS exposed oocytes. Taken together, these data suggest that the mechanisms governing oocyte mitophagy are fundamentally distinct from those governing somatic cell mitophagy and that the absence of mitophagy in the setting of HF/HS exposure contributes to the oocyte-to-blastocyst transmission of dysfunctional mitochondria.

Effects of single layer centrifugation (SLC) on bull spermatozoa prior to freezing on post-thaw semen characteristics.

Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen-thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species-specific colloid, Bovicoll. After centrifugation for 20min at 300g, the resulting sperm pellet was resuspended in OPTIXcell® (IMV Technologies, l'Aigle, France); the SLC-selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC-treated samples had a higher percentage of live, superoxide-positive spermatozoa than uncentrifuged samples (27.9±5.1% versus 21.7±6.7%; p=.03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9±8.2% versus 40.5±15.1%; p=.03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non-treated samples (42.0±8.5% versus 55.9±14.4%; p=.04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide-positive spermatozoa and live, hydrogen peroxide-negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC-selected spermatozoa compared to unselected samples.

Energy metabolism of two kinds of stromal fibroblasts differently fuel the development of oral squamous cell carcinoma

Background: Cancer is primarily a metabolic disease involving disturbances in cellular energy metabolism. “Reverse Warburg Effect” in adjacent stromal fibroblasts can be induced to feed cancer cells. Objectives: Develop a novel co-culture system to compare energy metabolism between normal oral fibroblasts (NOFs) and cancer-associated fibroblasts (CAFs), study their pathophysiological significance in microenvironment of oral squamous cell carcinoma (OSCC). Methods: CAFs and NOFs were both monolayer-cultured and co-cultured, respectively with normal oral keratinocytes (NOKs) and OSCC. Stained and analysed them by luminescent reader, flow cytometry, confocal microscope and Western blot for adenosine triphosphate (ATP) production, mitochondrial volume, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), lactate, caveolin-1 and monocarboxylate transporter 4 (MCT-4). Mean and standard deviation were plotted for 3 replicates from each condition. Findings: After co-cultured with OSCC, CAFs showed decreased ATP production, ROS, caveolin-1 and lower MMP, but mitochondrial volume and MCT-4 expression were increased. Interestingly, NOFs showed increased ATP production, ROS, lactate, MCT-4, but decreased MMP, mitochondrial volume and caveolin-1 after co-cultured with OSCC. Confocal work showed that mitochondrial transfer more easily happened from NOFs to OSCC. Conclusions: OSCC can induce “Reverse Warburg Effect” in both NOFs and CAFs, which present mitochondrial dysfunction and oxidative stress in different ways that fuel ATP production in OSCC. Interestingly, metabolic coupling and mitochondrial transfer occur between stromal fibroblasts and OSCC. As CAFs’ precursor, NOFs play a more significant role in invasive tumour front than CAFs, which dramatically increase aggressive behaviour of OSCC and associate with early tumour recurrence and metastasis.

Parkin-mediated mitophagy is downregulated in browning of white adipose tissue.

Browning of white adipose tissue (WAT) promotes increased energy expenditure through the action of uncoupling protein 1 (UCP1) and is an attractive target to promote weight loss in obesity. Lowering of mitochondrial membrane potential by UCP1 is uniquely beneficial in this context; in other tissues, reduced membrane potential promotes mitochondrial clearance via mitophagy. It is unknown how parkin-mediated mitophagy is regulated in beige adipocytes. The relationship between parkin expression and WAT browning was investigated in 3T3-L1 adipocytes and parkin-deficient male C57BL/6 mice in response to pharmacological browning stimuli. Rosiglitazone treatment in 3T3-L1 adipocytes promoted mitochondrial biogenesis, UCP1 expression, and mitochondrial uncoupling. Parkin expression was decreased and reduced mitochondrial-associated parkin, and p62 indicated a reduction in mitophagy activity. Parkin overexpression prevented mitochondrial remodeling in response to rosiglitazone. In CL 316,243-treated wild-type mice, decreased parkin expression was observed in subcutaneous inguinal WAT, where UCP1 was strongly induced. CL 316,243 treatment weakly induced UCP1 expression in the gonadal depot, where parkin expression was unchanged. In contrast, parkin-deficient mice exhibited robust UCP1 expression in gonadal WAT following CL 316,243 treatment. WAT browning was associated with a decrease in parkin-mediated mitophagy, and parkin expression antagonized browning of WAT.

Cyclophilin D Acetylation Regulates Cardiac Myocyte Differentiation

INTRODUCTION: Protein acetylation is an important regulator of cellular function. Acetylation of cyclophilin D (CyPD) at lysine 166 increases its ability to open the mitochondria permeability transition pore (PTP), which is an important regulator of mitochondrial metabolism and myocyte differentiation. HYPOTHESIS: Acetylation of CyPD controls PTP activity, mitochondrial structure and function, and myocyte differentiation in the developing heart. METHODS: We use immunoblotting and immunoprecipitation to determine the global protein acetylation state and that of CyPD during embryonic cardiac development in wild type (WT) and CyPD-null mice. We also re-expressed WT and mutant CyPD in CyPD-null embryonic and neonatal cardiac myocyte cultures to determine effects on mitochondrial structure and function and myocyte differentiation. RESULTS: Immunoblotting revealed that global protein acetylation was high in the embryo but fell during later development. In contrast, CyP-D was highly acetylated in the early embryonic heart, but this decreased dramatically during subsequent development. When re-expressed in cultures of CyPD-null myocytes, WT CyPD recapitulated the WT cell phenotype of fragmented mitochondrial structure, low mitochondrial membrane potential (Δψm), and decreased differentiation. Re-expression of a CyPD acetylation mimic mutant (K166Q) had similar or greater effects on these parameters than WT CyPD, while an inactivating (R96G) and de-acetylation mimic (K166R) CyPD mutant did not. CONCLUSION: These data suggest that myocyte differentiation during cardiac development is associated with a decrease in overall protein acetylation, perhaps because of the changes that occur in metabolism as the heart develops. However, specific acetylation of CyPD appears to be particularly important for maturation of mitochondrial function and regulation of cardiac myocyte differentiation.

GLUT3 upregulation promotes metabolic reprogramming associated with antiangiogenic therapy resistance.

Clinical trials revealed limited response duration of glioblastomas to VEGF-neutralizing antibody bevacizumab. Thriving in the devascularized microenvironment occurring after antiangiogenic therapy requires tumor cell adaptation to decreased glucose, with 50% less glucose identified in bevacizumab-treated xenografts. Compared with bevacizumab-responsive xenograft cells, resistant cells exhibited increased glucose uptake, glycolysis, 13C NMR pyruvate to lactate conversion, and survival in low glucose. Glucose transporter 3 (GLUT3) was upregulated in bevacizumab-resistant versus sensitive xenografts and patient specimens in a HIF-1α-dependent manner. Resistant versus sensitive cell mitochondria in oxidative phosphorylation-selective conditions produced less ATP. Despite unchanged mitochondrial numbers, normoxic resistant cells had lower mitochondrial membrane potential than sensitive cells, confirming poorer mitochondrial health, but avoided the mitochondrial dysfunction of hypoxic sensitive cells. Thin-layer chromatography revealed increased triglycerides in bevacizumab-resistant versus sensitive xenografts, a change driven by mitochondrial stress. A glycogen synthase kinase-3β inhibitor suppressing GLUT3 transcription caused greater cell death in bevacizumab-resistant than -responsive cells. Overexpressing GLUT3 in tumor cells recapitulated bevacizumab-resistant cell features: survival and proliferation in low glucose, increased glycolysis, impaired oxidative phosphorylation, and rapid in vivo proliferation only slowed by bevacizumab to that of untreated bevacizumab-responsive tumors. Targeting GLUT3 or the increased glycolysis reliance in resistant tumors could unlock the potential of antiangiogenic treatments.

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality.

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p<.05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2-deoxy-d-glucose, DOG). Furthermore, treatment with DOG also led to a dose-dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.

Oxidative status predicts quality in human mesenchymal stem cells

BackgroundHuman bone marrow-derived mesenchymal stem cells (MSC) are adult progenitor cells with great potential for application in cell-based therapies. From a cell-based therapy perspective, there are two limitations to MSC use: (1) these therapies require large numbers of cells, and long-term expansion of MSC in vitro promotes replicative senescence; and (2) patient variability is a challenge for defining MSC quality standards for transplantation. This study aimed to determine whether low or high oxidative status of MSC correlate with changes in cell expansion and differentiation potentials.MethodsWe investigated functional aspects of mitochondria, such as cell metabolic activity indicators and expression of antioxidant enzymes. Furthermore, we tested if senescence-induced changes in oxidative status of MSC could be counteracted by methylene blue (MB), an alternative mitochondrial electron transfer known to enhance cell bioenergetics.ResultsMSC isolated from donors of the same age showed distinctive behavior in culture and were grouped as weak (low colony-forming units (CFU) and a short life in vitro) and vigorous MSC (high CFU and a long life in vitro). In comparison to weak MSC, vigorous MSC had oxidative status characterized by lower mitochondrial membrane potential, lower mitochondrial activity, and fewer reactive oxygen species production, as well as reduced mitochondrial biogenesis. Vigorous MSC had a significantly higher expansion potential compared to weak MSC, while no differences were observed during differentiation. MB treatment significantly improved expansion and differentiation potential, however only in vigorous MSC.ConclusionsTogether, these results demonstrate the importance of mitochondrial function in MSC in vitro, and that cells with low oxidative status levels are better candidates for cell-based therapies.

Ultrasound hyperthermia induces apoptosis in head and neck squamous cell carcinoma: an in vitro study

BackgroundHyperthermia is considered an efficient complement in the treatment of head and neck squamous cell carcinoma (HNSCC). Hyperthermia induces cell apoptosis in a temperature- and time-dependent manner. However, the molecular mechanism of hyperthermia remains unclear. The aim of this study was to investigate the mechanism of apoptosis induced by ultrasound hyperthermia in HNSCC cell lines HN-30 and HN-13.Material and MethodsWe examined the dynamic changes of early apoptosis and secondary necrosis in HN-30 and HN-13 cells treated by hyperthermia at 42°C for 10 min. We further examined mitochondrial membrane potential in vitro by ultrasound hyperthermia for different heating temperatures (38-44°C, 10 min) and heating times (42°C, 10-50 min). After heating by ultrasound at 42°C for 10 min, the apoptosis index achieved its highest level at 8 h after treatment, decreased rapidly and remained constant at a reduced level at 12 h.ResultsThe level of secondary necrosis increased with the level of early apoptosis but remained at a higher level until 14 h. The level of secondary necrosis correlated with the level of early apoptosis (HN-13: r=0.7523, P=0.0030; HN-30: r=0.6510, P=0.016). The fractions of cells with low mitochondrial membrane potential (Δψ) in the heating-temperature grads group and heating-time grads group decreased significantly over time. Therefore, HN-30 and HN-13 cells developed apoptosis after ultrasound hyperthermia treatment with decreases in the mitochondrial transmembrane potential level.ConclusionsUltrasound hyperthermia induces apoptosis in HN-30 and HN-13 cells, possibly via the mitochondrial caspase pathway. Key words:Head and neck squamous cell carcinoma, apoptosis, mitochondrial membrane potential, ultrasound hyperthermia.

Migration and invasion of drug-resistant lung adenocarcinoma cells are dependent on mitochondrial activity.

A small proportion of cancer cells have stem-cell-like properties, are resistant to standard therapy and are associated with a poor prognosis. The metabolism of such drug-resistant cells differs from that of nearby non-resistant cells. In this study, the metabolism of drug-resistant lung adenocarcinoma cells was investigated. The expression of genes associated with oxidative phosphorylation in the mitochondrial membrane was negatively correlated with the prognosis of lung adenocarcinoma. Because the mitochondrial membrane potential (MMP) reflects the functional status of mitochondria and metastasis is the principal cause of death due to cancer, the relationship between MMP and metastasis was evaluated. Cells with a higher MMP exhibited greater migration and invasion than those with a lower MMP. Cells that survived treatment with cisplatin, a standard chemotherapeutic drug for lung adenocarcinoma, exhibited increased MMP and enhanced migration and invasion compared with parental cells. Consistent with these findings, inhibition of mitochondrial activity significantly impeded the migration and invasion of cisplatin-resistant cells. RNA-sequencing analysis indicated that the expression of mitochondrial complex genes was upregulated in cisplatin-resistant cells. These results suggested that drug-resistant cells have a greater MMP and that inhibition of mitochondrial activity could be used to prevent metastasis of drug-resistant lung adenocarcinoma cells.