Mitochondrial Metabolic Activity Research Articles

Citrate Synthase Is a NovelIn VivoMatrix Metalloproteinase-9 Substrate That Regulates Mitochondrial Function in the Postmyocardial Infarction Left Ventricle

To evaluate the role of matrix metalloproteinase (MMP)-9 deletion on citrate synthase (CS) activity postmyocardial infarction (MI). We fractionated left ventricle (LV) samples using a differential solubility-based approach. The insoluble protein fraction was analyzed by mass spectrometry, and we identified CS as a potential intracellular substrate of MMP-9 in the MI setting. CS protein levels increased in the insoluble fraction at day 1 post-MI in both genotypes (p<0.05) but not in the noninfarcted remote region. The CS activity decreased in the infarcted tissue of wild-type (WT) mice at day 1 post-MI (p<0.05), but this was not observed in the MMP-9 null mice, suggesting that MMP-9 deletion helps to maintain the mitochondrial activity post-MI. Additionally, inflammatory gene transcription was increased post-MI in the WT mice and attenuated in the MMP-9 null mice. MMP-9 cleaved CS in vitro, generating an ∼20 kDa fragment. By applying a sample fractionation and proteomics approach, we were able to identify a novel MMP-9-related altered mitochondrial metabolic activity early post-MI. Our data suggest that MMP-9 deletion improves mitochondrial function post-MI.

The Association of Mitochondrial Potential and Copy Number with Pig Oocyte Maturation and Developmental Potential

ATP is critical for oocyte maturation, fertilization, and subsequent embryo development. Both mitochondrial membrane potential and copy number expand during oocyte maturation. In order to differentiate the roles of mitochondrial metabolic activity and mtDNA copy number during oocyte maturation, we used two inhibitors, FCCP (carbonyl cyanide p-(tri-fluromethoxy)phenyl-hydrazone) and ddC (2’3-dideoxycytidine), to deplete the mitochondrial membrane potential (Δφm) and mitochondrial copy number, respectively. FCCP (2000 nM) reduced ATP production by affecting mitochondrial Δφm, decreased the mRNA expression of Bmp15 (bone morphogenetic protein 15), and shortened the poly(A) tails of Bmp15, Gdf9 (growth differentiation factor 9), and Cyclin B1 transcripts. FCCP (200 and 2000 nM) also affected p34cdc2 kinase activity. By contrast, ddC did not alter ATP production. Instead, ddC significantly decreased mtDNA copy number (P < 0.05). FCCP (200 and 2000 nM) also decreased extrusion of the first polar body, whereas ddC at all concentrations did not affect the ability of immature oocytes to reach metaphase II. Both FCCP (200 and 2000 nM) and ddC (200 and 2000 µM) reduced parthenogenetic blastocyst formation compared with untreated oocytes. However, these inhibitors did not affect total cell number and apoptosis. These findings suggest that mitochondrial metabolic activity is critical for oocyte maturation and that both mitochondrial metabolic activity and replication contribute to the developmental competence of porcine oocytes.

Effective Targeting Of The P53/MDM2 Axis In Preclinical Models Of Infant MLL-Rearranged Acute Lymphoblastic Leukemia

IntroductionWhile the overall cure rate for the most common pediatric cancer, acute lymphoblastic leukemia (ALL) now approaches 90%, infants (<12 months) diagnosed with ALL harboring translocations in the mixed-lineage leukemia oncogene (infant MLL-ALL) experience shorter remission duration and a significantly reduced likelihood of survival (∼50%). Therefore, new treatments that can be incorporated into conventional chemotherapy regimens to extend patient remission and improve survival are urgently required. Mutations in the p53 tumor suppressor are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be of therapeutic benefit. Nutlin cis-imidazole molecules selectively inhibit p53-MDM2 binding, resulting in activation of the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenograft models. MethodsIn vitro cytotoxicity was assessed by mitochondrial metabolic activity assay (Alamar blue) following 48h drug exposures. P53 protein levels and subcellular distribution were assessed by immunoblotting. Patient-derived xenografts were established from infant MLL-ALL, B-cell precursor (BCP)-ALL, or T-lineage ALL (T-ALL) bone marrow or peripheral blood (PB) biopsies in immune-deficient (NOD/SCID or NSG) mice, and their gene expression profiles generated using Illumina Human Ref-12 Expression BeadChips. Engraftment and drug responses were assessed by enumeration of the proportion of human versus mouse CD45+ cells in the PB. Mice with established disease received vehicle, RG7112 (100 mg/kg daily x 5 p.o.), a combination of vincristine (0.15 mg/kg once i.p.) dexamethasone (5 mg/kg daily x 5 i.p.) and L-asparaginase (1,000 IU/kg daily x 5 i.p.) (VXL), or RG7112 plus VXL. Anti-leukemic efficacy was assessed using an objective response measure modeled after the clinical setting, as well as the median event-free survival (EFS) of treated or control groups from treatment initiation. Therapeutic enhancement was considered to occur when the RG7112/VXL combination significantly extended mouse EFS compared with that of both of the RG7112 and VXL treated groups. ResultsUnsupervised hierarchical clustering of gene expression profiles revealed that the MLL-ALL (n=9), BCP-ALL (n=7) and T-ALL (n=13) xenografts clustered according to leukemia subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA gene family, were significantly upregulated in MLL-ALL xenografts. The specificity of RG7112 was validated by cytotoxicity assays against leukemia cell lines of known p53 status; p53 wild-type cell lines (RS4;11, IC50 1.4 µM; NALM-6, IC50 3.0 µM) were markedly more sensitive than those with mutant p53 (CEM, IC50 >10 µM; JURKAT, IC50 >10 µM). The in vitro sensitivity of BCP-ALL (n=3) and infant MLL-ALL (n=4) xenografts was consistent with wild-type p53 status, with IC50s of 0.11 - 2.2 µM. Exposure of ALL xenograft cells to nutlin-3 (10 µM, 6h) caused marked p53 up-regulation and nuclear translocation. Since we had previously shown that RG7112 administered as a single agent for 14 days induced significant regressions [Complete Responses (CRs) or greater] in 7/7 infant MLL-ALL xenografts in vivo, we assessed its efficacy in a 5-day combination treatment with an induction-type regimen (VXL) against two infant MLL-ALL xenografts (MLL-5 and MLL-14). The RG7112/VXL combination caused a Partial Response in MLL-5 compared with Progressive Disease for both RG7112 and VXL. The efficacy of RG7112/VXL was even more pronounced against MLL-14, causing a Maintained CR compared with CRs for both RG7112 and VXL, which met the criteria for Therapeutic Enhancement (the median EFS of RG7112/VXL-treated mice, 65.0 days, was significantly greater, P< 0.0001, than that of RG7112, 22.2 days, and VXL, 28.5 days). ConclusionsRG7112 induces significant regressions in a high proportion of infant MLL-ALL xenografts and enhances the efficacy of an induction-type regimen. The utility of targeting the p53-MDM2 axis in combination with established drugs for the clinical management of infant MLL-ALL warrants further investigation.This study was supported by NCI NO1CM42216. The authors thank Roche Pharmaceuticals, Inc., for providing RG7112. Disclosures:No relevant conflicts of interest to declare.

P.11.5 PMO-mediated dystrophin exon 23 skipping restores mitochondrial function in the mdx mouse heart

Approximately 20% of boys with Duchenne Muscular Dystrophy will die of dilated cardiomyopathy. The cardiomyopathy is characterised by disrupted structure and function of cardiac muscle cells and reduced energy production. However, the mechanisms responsible for the altered energy metabolism have been poorly understood. We have previously sought to identify the mechanisms for metabolic inhibition in mdx mouse cardiomyopathy. Calcium influx through the L-type Ca2+ channel (also known as the dihydropyridine receptor) in cardiac myocytes is essential for contraction. Calcium is also important for the regulation of mitochondrial function and production of ATP that is required to meet the energy demands of the heart. We have shown that the L-type Ca2+ channel can regulate mitochondrial function and metabolic activity in cardiac myocytes. In mdx heart, the communication between the L-type Ca2+ channel and the mitochondria is altered as a result of disruption of the cytoskeletal architecture. This contributes to metabolic inhibition in the mdx heart. We demonstrate that treatment of mdx mice with a phosphorodiamidate morpholino oligomer, designed to induce skipping of dystrophin exon 23, “restored” the increase in mitochondrial membrane potential in mdx cardiomyocytes after activation of the L-type Ca2+ channel with the dihydropyridine receptor agonist BayK (−). These results confirm that metabolic inhibition occurs as a result of the absence of dystrophin, and oligomer therapy may be able to normalise metabolic activity and restore contractility in mdx mouse heart.

Combined oral contraceptives affect liver mitochondrial activity

Objectives To examine liver mitochondrial function in women using combined oral contraceptives (COCs) containing ethinylestradiol.Methods A breath test after oral administration of 1 mg/kg 13C-alpha-ketoisocaproic acid (13C-KICA) and 20 mg/kg L-leucine was performed twice: (i) in 15 women on day 14, 15, 16, 17 or 18 of COC intake, and between day 1 and 5 of the withdrawal bleeding; and (ii) in 15 regularly menstruating females not taking hormonal contraceptives: during the luteal phase, between the 18th and the 22nd day of the cycle, and again between day 1 and 5 of the menstruation.Results In women on COCs the maximum 13C elimination in breath air (Dmax) was higher (26.8 ± 1.6%/h) than during withdrawal bleeding (23.5 ± 1.2%/h; p = 0.012). The time to reach the Dmax was similar on the two study days: 33.3 ± 2.4 min during the phase of pill intake vs. 37.0 ± 2.5 min during the pill-free interval. The one-hour cumulative breath 13C elimination was greater after two weeks of COC intake than during the withdrawal bleeding: 17.49 ± 1.03% vs. 15.32 ± 0.85% (p = 0.024). In the control group no menstrual cycle phase-dependent fluctuations in the results of the 13C-KICA breath test were observed.Conclusion The metabolism of 13C-alpha-ketoisocaproic acid augments during the intake of COCs containing ethinylestradiol, reflecting enhanced liver mitochondrial metabolic activity.

Loss of caspase-2 accelerates age-dependent alterations in mitochondrial production of reactive oxygen species

Mitochondria are known to be a major source and target of oxidative stress. Oxidative stress increases during aging and is suggested to underlie in part the aging process. We have previously documented an increase in endogenous caspase-2 (casp2) activity in hepatocytes obtained from old (28 months) vs. young mice (5 months). More recently, we have shown that casp2 is activated by oxidative stress and is critical for mitochondrial oxidative stress-induced apoptosis. Since casp2 appears integral to mitochondrial oxidative stress-induced apoptosis, in this study we determined whether loss of casp2 altered the production of mitochondrial reactive oxygen radicals (mROS) as a function of age in intact living hepatocytes. To stimulate mitochondrial metabolic activity, we added a mixture of pyruvate and glutamate to hepatocytes while continuously monitoring endogenous mROS production in the presence or absence of rotenone and/or antimycin A. Our data demonstrate that mROS production and neutralization are compromised in hepatocytes of old mice. Interestingly, casp2 deficient hepatocytes from middle age mice (12 months) had similar mROS neutralization kinetics to those of hepatocytes from old WT mice. Rotenone had no effect on mROS metabolism, whereas antimycin A significantly altered mROS production and metabolism in an age-dependent fashion. Our results indicate that: (1) hepatocytes from young and old mice respond differently to dysfunction of the mitochondrial electron transport chain; (2) age-dependent alterations in mROS metabolism are likely regulated by complex III; and (3) absence of casp2 accelerates age-dependent changes in terms of pyruvate/glutamate-induced mROS metabolism.

Essential Role of Thioredoxin 2 in Mitigating Oxidative Stress in Retinal Epithelial Cells

The retina is constantly subjected to oxidative stress, which is countered by potent antioxidative systems present in retinal pigment epithelial (RPE) cells. Disruption of these systems leads to the development of age-related macular degeneration. Thioredoxin 2 (Trx2) is a potent antioxidant, which acts directly on mitochondria. In the present study, oxidative stress was induced in the human RPE cell line (ARPE-19) using 4-hydroxynonenal (4-HNE) or C2-ceramide. The protective effect of Trx2 against oxidative stress was investigated by assessing cell viability, the kinetics of cell death, mitochondrial metabolic activity, and expression of heat shock proteins (Hsps) in Trx2-overexpressing cell lines generated by transfecting ARPE cells with an adeno-associated virus vector encoding Trx2. We show that overexpression of Trx2 reduced cell death induced by both agents when they were present in low concentrations. Moreover, early after the induction of oxidative stress Trx2 played a key role in the maintenance of the cell viability through upregulation of mitochondrial metabolic activity and inhibition of Hsp70 expression.

Integration of clinical data with a genome-scale metabolic model of the human adipocyte.

We evaluated the presence/absence of proteins encoded by 14 077 genes in adipocytes obtained from different tissue samples using immunohistochemistry. By combining this with previously published adipocyte-specific proteome data, we identified proteins associated with 7340 genes in human adipocytes. This information was used to reconstruct a comprehensive and functional genome-scale metabolic model of adipocyte metabolism. The resulting metabolic model, iAdipocytes1809, enables mechanistic insights into adipocyte metabolism on a genome-wide level, and can serve as a scaffold for integration of omics data to understand the genotype-phenotype relationship in obese subjects. By integrating human transcriptome and fluxome data, we found an increase in the metabolic activity around androsterone, ganglioside GM2 and degradation products of heparan sulfate and keratan sulfate, and a decrease in mitochondrial metabolic activities in obese subjects compared with lean subjects. Our study hereby shows a path to identify new therapeutic targets for treating obesity through combination of high throughput patient data and metabolic modeling.

Low concentrations of Rhodamine-6G selectively destroy tumor cells and improve survival of melanoma transplanted mice

Rhodamine-6G is a fluorescent dye binding to mitochondria, thus reducing the intact mitochondria number and inhibiting mitochondrial metabolic activity. Resultantly, the respiratory chain functioning becomes blocked, the cell "suffocated" and eventually destroyed. Unlike normal cells, malignant cells demonstrate a priori reduced mitochondrial numbers and aberrant metabolism. Therefore, a turning point might exist, when Rhodamine-induced loss of active mitochondria would selectively destroy malignant, but spare normal cells. Various malignant vs. non-malignant cell lines were cultured with Rhodamine-6G at different concentrations. In addition, C57Bl mice were implanted with B16-F10 melanoma and treated with Rhodamine-6G at different dosage/time regimens. Viability and proliferation of cultured tumor cells were time and dose-dependently inhibited, up to 90%, by Rhodamine-6G, with profound histological signs of cell death. By contrast, inhibition of normal control cell proliferation hardly exceeded 15-17%. Melanoma-transplanted mice receiving Rhodamine-6G demonstrated prolonged survival, improved clinical parameters, inhibited tumor growth and metastases count, compared to their untreated counterparts. Twice-a-week 10-6M Rhodamine-6G regimen yielded the most prominent results. We conclude that malignant, but not normal, cells are selectively destroyed by low doses of Rhodamine-6G. In vivo, such treatment selectively suppresses tumor progression and dissemination, thus improving prognosis. We suggest that selective anti-tumor properties of Rhodamine-6G are based on unique physiologic differences in energy metabolism between malignant and normal cells. If found clinically relevant, low concentrations of Rhodamine-6G might be useful for replacing, or backing up, more aggressive nonselective chemotherapeutic compounds.

The low doses effect of experimental zearalenone (ZEN) intoxication on the presence of Ca2+ in selected ovarian cells from pre-pubertal bitches

The objective of this study was to determine the effect of 42-day ZEN intoxication on the presence of Ca2+ in selected ovarian cells from beagle bitches, using the potassium pyroantimonate (PPA) method. Samples were collected from 30 clinically healthy, pre-pubertal, genetically homogeneous animals. The bitches were divided into three groups of 10 animals each: experimental group I--50 microg ZEN/kg BW (100% NOAEL) administered once daily per os; experimental group II--75 microg ZEN/kg BW (150% NOAEL) administered once dailyper os; control group--placebo containing no ZEN administered per os. An electron microscopic analysis revealed that cells died due to apoptosis, depending on the ZEN dose and the type of cells exposed to intoxication. Lower ZEN doses led to apoptosis-like changes in the cells. Cell death was a consequence of excess Ca2+ accumulation in the mitochondria, followed by cell dysfunction and a decrease in or the absence of mitochondrial metabolic activity in oocytes, follicle cells and interstitial cells in experimental bitches.

Seasonal change in mitochondrial function and metabolic enzyme activity of different populations of the mud crab, Scylla paramamosain, from China

Over past decades, the farming of mud crab Scylla paramamosain has expanded rapidly in China. In our previous experiments, the results showed that the mud crab S. paramamosain in China could be divided into northern and southern populations and these two populations had differences in the adaptive capacity to cultivation environmental conditions. In order to verify this, the present study measured and compared the mitochondrial function and metabolic enzyme activity of these two populations during seasonal acclimatization. It was shown that the mitochondrial respiration rates in the gill, hepatopancreas and muscle of the northern population were significantly higher than those of the southern population in winter, spring and summer although the differences in summer were less pronounced than in winter and spring. In winter and spring, both state 2 and 3 mitochondrial respiration rates measured using either succinate or pyruvate+malate as the substrate in the gill of the northern population were consistently significantly higher than those of the southern population. The measurement of metabolic enzyme activity during different seasons showed that for the mitochondrial cytochrome C oxidase in the gill, hepatopancreas and muscle, the differences in activity between the northern and southern populations were in general consistent with those of the mitochondrial function. For example, the mitochondrial cytochrome C oxidase activities in the gill of the northern population were significantly higher than those of the southern population in winter, spring and summer. However, activity of lactate dehydrogenase, pyruvate kinase and hexokinase in the muscle were slightly different between the northern and southern populations. Only in summer the lactate dehydrogenase and pyruvate kinase activities in the muscle of the southern population were significantly higher than those of the northern population. In summary, the results of the present study showed that compared to the southern population, the northern population had a better compensation mechanism in mitochondrial metabolite level during cooler seasons, which might indicate that the northern population adapts better to low temperature than the southern population. Therefore, the temperature for the farming of the southern population and northern population should be different.

Effects of Red Light-emitting Diode Irradiation on Dental Pulp Cells

Light irradiation activates a range of cellular processes in a variety of cell types, including stem cells, and can promote tissue repair. This study investigated the effects of light-emitting diode (LED) exposure on dental pulp cells (DPCs). Dose response analysis at 20-second intervals up to 120 seconds demonstrated that a LED array emitting 653-nm red light stimulated significantly increased cell growth at 3 and 7 days post-irradiation with 40 (149 mJ/cm2) and 60 (224 mJ/cm2) seconds of radiant exposure. Double-dosing cells at days 1 and 4 of a 7-day culture period with 60-second (224 mJ/cm2) LED exposure significantly increased cell growth compared with a single dosing regime. BrdU analysis demonstrated significantly increased proliferation rates associated with significantly increased ATP, nitric oxide (NO), and mitochondrial metabolic activity. LED-stimulated NO levels were not reduced by inhibition of NO-synthase activity. Light exposure also rescued the inhibition of mitochondrial dysfunction and increased levels of in vitro mineralization compared with control. Media exchange experiments indicated that autocrine signaling was not likely responsible for red-light-induced DPC activity. In conclusion, data analysis indicated that 653-nm LED irradiation promoted DPC responses relevant to tissue repair, and this is likely mediated by increased mitochondrial activity.

Effects of an Experimental Calcium Aluminosilicate Cement on the Viability of Murine Odontoblast-like Cells

IntroductionQuick-setting calcium aluminosilicate cement with improved washout resistance is a potential substitute for calcium silicate cements in endodontics. This study examined the effect of an experimental calcium aluminosilicate cement (Quick-Set; Primus Consulting, Bradenton, FL) on the viability of odontoblast-like cells. MethodsThe biocompatibility of Quick-Set and white ProRoot MTA (WMTA; Dentsply Tulsa Dental Specialties, Tulsa, OK) cements and their eluents was evaluated using a murine dental papilla–derived odontoblast-like cell line (MDPC-23); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to examine the effects of the 2 hydraulic cements on mitochondrial metabolic activity. Flow cytometry and confocal laser scanning microscopy were used to identify the effects of the 2 cements on cell death–induced plasma membrane permeability to fluorescent dyes and DNA stains. ResultsAfter the first week of immersion in culture medium, Quick-Set and WMTA were more cytotoxic than the Teflon-negative control (P < .05), and the cells exhibited more apoptosis/necrosis than Teflon (P < .05). After the second week of immersion, the 2 cements were as biocompatible as Teflon (P > .05), with cells exhibiting minimal apoptosis/necrosis. Eluents from the set cements at 1:1 dilution were significantly more cytotoxic that eluents at 1:10 or 1:100 dilution (P < .05). ConclusionsQuick-Set and WMTA exhibited similar cytotoxicity profiles. They possess negligible in vitro toxicologic risks after time-dependent elution of toxic components.

DJ-1 Based Peptide as a Novel Neuroprotective Strategy for Parkinson's Disease (SC02.003)

Objective: To explore a possible neuroprotective treatment for Parkinson9s disease (PD) based on DJ-1. Background Loss-of-function mutations in DJ-1 cause early-onset familial PD. Abnormal DJ-1 levels in the plasma, CSF and brains of sporadic PD patients imply that DJ-1 might be involved in sporadic PD. Studies demonstrated that DJ-1 defends against oxidative stress. Design/Methods: Based on a thorough bio-informatic survey and knowledge acquired through our studies on DJ-1, a series of DJ-1-based peptides was designed. The protective effects of these peptides were tested on cell-lines and on primary cultures obtained from mice brains. Mitochondrial damage (by MTT assay), metabolic activity (by Alamar blue method), cell toxicity (by LDH detection), and viability (by Hoechst staining), were examined with exposure to neurotoxic insults. In vivo effects were tested using 6-hydroxydopamine and MPTP mice models, employing c57/bl and DJ-1 knockout mice. Behavioral, biochemical and histological parameters in mice treated with the chosen peptide (named ND-13) were compared to control peptides and vehicle. Statistical analysis was done using ANOVA with p Results: ND-13 was shown to protect cultured neurons against oxidative and neurotoxic insults. In vivo tests with intravenous or subcutaneous injections of ND-13 demonstrated protection against 6-hydroxydopamine striatal lesioning, as indicated by behavioral, biochemical and histochemical measurements. Subcutaneous injections of ND-13, one hour before MPTP treatments, given on five consecutive days, restored dopamine and tyrosine hydroxylase levels. A series of 6-hydroxydopamine and MPTP experiments on DJ-1 knockout mice demonstrated similar results. Conclusions: DJ-1-related peptide demonstrated significant neuroprotective abilities in well established models of PD. The peptide may serve as a potential novel strategy to slow the neurodegenerative process and alter the disease course, thereby providing long term benefit for PD patients. Supported by: The Israeli Science Foundation (ISF) and the Norma and Alan Aufzien chair for the research of Parkinson9s disease, Tel Aviv University, Israel. Disclosure: Dr. Lev has nothing to disclose. Dr. Barhum has nothing to disclose. Dr. Ben-Tzur has nothing to disclose. Dr. Melamed has nothing to disclose. Dr. Offen has nothing to disclose.

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.

Abstract 1758: A podophyllotoxin derivative, Adva-27a, is more potent at killing multi-drug resistant breast cancer cells than etoposide

Abstract A novel non-ester GEM-difluorinated C-glycoside derivative of podophyllotoxin, Adva-27a, had been designed and synthesized. The growth inhibitory properties of this drug compared to the currently used Topoisomerase II inhibitor etoposide was evaluated in MCF-7 breast cancer cells and in a multi-drug resistant (MDR) variant of this cell line. These cells were cultured in the presence of the drugs at different concentrations (0.1-100μM) for various times (1-9 days) and analyzed for viability using a mitochondrial metabolic activity dye, Alamar blue versus solvent (DMSO) treated cells. The MCF-7 cells were sensitive to Adva-27a and etoposide, resulting in an IC50 of less than 10μM for etoposide and 20μM for Adva-27a, while the IC50 for the MDR MCF-7 cells was 25μM with Adva-27a, but greater than 100μM for etoposide. The timing for killing by Adva-27a of the MDR MCF-7 cells appeared significantly different than for the MCF-7 parent cells. In this case, 100μM drug resulted in less than 50% of viability at day 3 for the MDR MCF-7 cells, while the same concentration of drug required 5-6 days to cause the same level of killing of the parent cells. To determine the mechanism of action of the drug, we analyzed the Adva-27a treated cells by flow cytometry using propidium iodide staining. We found that after 24 hours with 30μM of the drug, both MCF-7 and the MDR MCF-7 cells were accumulating in the G2/M phase of the cell cycle. Both cells are similarly killed after 7 days by this concentration of the drug. Etoposide is known to cause accumulation of cells at a similar phase, implying the Adva-27a has a similar drug target. Thus, the mechanism for the difference in sensitivity of the MDR MCF-7 cells for Adva-27a relative to etoposide is not yet understood, but will be explored in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1758. doi:1538-7445.AM2012-1758

Depression of mitochondrial metabolism by downregulation of cytoplasmic deacetylase, HDAC6

Mitochondria perform multiple functions critical to the maintenance of cellular homeostasis. Here we report that the downregulation of histone deacetylase 6 (HDAC6) causes a reduction in the net activity of mitochondrial enzymes, including respiratory complex II and citrate synthase. HDAC6 deacetylase and ubiquitin-binding activities were both required for recovery of reduced mitochondrial metabolic activity due to the loss of HDAC6. Hsp90, a substrate of HDAC6, localizes to mitochondria and partly mediates the regulation of mitochondrial metabolic activity by HDAC6. Our finding suggests that HDAC6 regulates mitochondrial metabolism and might serve as a cellular homeostasis surveillance factor.

Evidence for Altered Communication Between the L-Type Ca2+ Channel and Mitochondria in a Model of Cardiomyopathy

Progression of cardiac hypertrophy to failure and development of many cardiomyopathies involves myocyte remodeling, disorganisation of cytoskeletal proteins and reduced energy metabolism. The mechanisms by which cytoskeletal disruption leads to mitochondrial dysfunction are poorly understood.Calcium influx through the L-type Ca2+ channel (LTCC) is a requirement for contraction in the heart. The LTCC can influence mitochondrial superoxide production, NADH production and metabolic activity in a calcium-dependent manner. Activation of the channel can also increase mitochondrial membrane potential (Ψm) in a calcium-independent manner. This response is dependent upon the cytoskeleton. We hypothesized that disruption of normal cytoskeletal architecture will result in altered communication between the LTCC and mitochondria. We investigated this hypothesis in the murine model of Duchenne Muscular Dystrophy(mdx). Myocytes from 8 week old mdx mice that exhibit disorganised cytoskeletal protein networks but not yet overt cardiomyopathy, demonstrated significantly slower LTCC inactivation rate (26.2±1.8ms, n=13 vs 21.1±1.3ms, n=16; p<0.05) and significantly greater calcium influx (681±40nM, n=7 vs 432±51nM, n=5). However activation of the LTCC did not increase Ψm (n=6) or metabolic activity (measured as formation of formazan from tetrazolium salt) in mdx myocytes (n=8). Application of 4,4′diisothiocyano-2,2′-stilbenedisulfonic acid, that blocks anion transport mimicked the response of BayK(-) in control myocytes (n=4) and “restored” the increase in Ψm in mdx myocytes (n=4).The activities of the mitochondrial respiratory complexes were normal in mitochondria isolated from 8 week old mdx hearts and from 40 week old mdx hearts that had developed cardiomyopathy. We conclude that the communication between the L-type Ca2+ channel and mitochondria is altered in the dystrophin-deficient cardiomyocyte. This appears to involve an alteration in the association between the channel protein, actin filaments and mitochondrial anion transport and precedes the development of cardiomyopathy.

Dietary Fish Oil Promotes Colonic Apoptosis and Mitochondrial Proton Leak in Oxidatively Stressed Mice

An alteration of mitochondrial function can result in disruption of redox homeostasis and is associated with abnormal cancer cell growth. Manganese superoxide dismutase (SOD2) and glutathione peroxidase 4 (Gpx4) are two of the most important antioxidant defense enzymes that protect cells against oxidative stress. We had previously shown that n-3 polyunsaturated fatty acids (PUFA) promote colonocyte apoptosis, a marker of colon cancer risk, in part by enhancing phospholipid oxidation. To elucidate the mechanisms regulating oxidative stress-induced apoptosis in vivo, we fed heterozygous SOD2(Het), Gpx4(Het), and transgenic Gpx4(Tg) mice diets containing either 15% corn oil by weight (CO, enriched in n-6 PUFA) or 3.5% CO + 11.5% fish oil (FO, enriched in n-3 PUFA) for 4 weeks. Our data showed that (i) genetic predeposition to oxidative stress facilitates apoptosis in the mouse colon (Gpx4(Het) > SOD2(Het) > Wt > Gpx4(Tg)), (ii) dietary n-3 PUFA have an additive effect on the induction of apoptosis in Gpx4(Het) and SOD2(Het) mice; and (iii) dietary n-3 PUFA reverse the phenotype in oxidatively protected Gpx4(Tg) mice by elevating apoptosis to a level observed in wild-type (Wt; control) animals. Complimentary experiments examining colonic mitochondrial bioenergetic profiles indicate that FO-fed mice exhibit a significantly (P < 0.05) increased respiration-induced proton leak relative to control CO treatment. This finding was consistent with a loss of membrane potential in response to chronic oxidative stress and supports the contention that n-3 PUFA alter mitochondrial metabolic activity, thereby enhancing apoptosis and reducing colon cancer risk.

The novel phloroglucinol PMT7 kills glycolytic cancer cells by blocking autophagy and sensitizing to nutrient stress

The switch from oxidative phosphorylation to glycolytic metabolism results in cells that generate fewer reactive oxygen species (ROS) and are resistant to the intrinsic induction of apoptosis. As a consequence, glycolytic cancer cells are resistant to radiation and chemotherapeutic agents that rely on production of ROS or intrinsic apoptosis. Further, the level of glycolysis correlates with tumor invasion, making glycolytic cancer cells an important target for new therapy development. We have synthesized a novel redox-active quinone phloroglucinol derivative, PMT7. Toxicity of PMT7 was in part due to loss of mitochondrial membrane potential in treated cells with subsequent loss of mitochondrial metabolic activity. Mitochondrial gene knockout ρ0 cells, a model of highly glycolytic cancers, were only half as sensitive as the corresponding wild-type cells and metabolic pathways downstream of MET were unaffected in ρ0 cells. However, PMT7 toxicity was also due to a block in autophagy. Both wild-type and ρ0 cells were susceptible to autophagy blockade, and the resistance of ρ0 cells to PMT7 could be overcome by serum deprivation, a situation where autophagy becomes necessary for survival. The stress response class III deacetylase SIRT1 was not significantly involved in PMT7 toxicity, suggesting that unlike other chemotherapeutic drugs, SIRT1-mediated stress and survival responses were not induced by PMT7. The dependence on autophagy or other scavenging pathways makes glycolytic cancer cells vulnerable. This can be exploited by induction of energetic stress to specifically sensitize glycolytic cells to other stresses such as nutrient deprivation or potentially chemotherapy.