Loss Of ATP Research Articles

Dehydrocostus lactone prevents mitochondrial dysfunction in osteoblastic MC3T3-E1 cells

The dried root of Saussurea lappa Clarke (Compositae) has been used as a traditional medicine. Dehydrocostus lactone is one of the main bioactive constituents of this medicinal plant. In the present study, the protective effect of dehydrocostus lactone against antimycin A (an inhibitor of mitochondrial complex III)-induced cytotoxicity was investigated in osteoblastic MC3T3-E1 cells. Pre-treatment with dehydrocostus lactone prior to antimycin A exposure significantly prevented mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, cytochrome c release, intracellular calcium elevation and potassium loss, and reactive oxygen species production induced by antimycin A. These results suggest that dehydrocostus lactone protects osteoblastic MC3T3-E1 cells from antimycin A-induced cell damage through the improved mitochondrial function.

Protective effect of apocynin on antimycin A‐induced cell damage in osteoblastic MC3T3‐E1 cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH-oxidase. In the present study, we investigated the protective effects of apocynin on antimycin A (AMA)-induced toxicicy in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to AMA caused significant cell viability loss, as well as mitochondrial membrane potential (MMP) dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca2+]i) elevation and oxidative stress. Pretreatment with apocynin prior to AMA exposure significantly reduced AMA-induced cell damage by preventing MMP dissipation, complex IV inactivation, ATP loss, [Ca2+]i elevation and oxidative stress. These results suggest that apocynin has a protective effect against AMA-induced cell damage by its antioxidant effects and the attenuation of mitochondrial dysfunction. Apocynin also induced the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by AMA. All these data indicate that apocynin may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders.

Abstract 5369: Prostate cancer radiosensitization through PARP-1 hyperactivation

Abstract The clinical experimental agent, beta-lapachone (Arq 501), can act as a potent radiosensitizer in vitro through an unknown mechanism. In this study, we analyzed the mechanism to determine whether beta-lapachone may warrant clinical evaluation as a radiosensitizer. Beta-lapachone killed prostate cancer cells by NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolic bioactivation, triggering a massive induction of reactive oxygen species (ROS), irreversible DNA single strand breaks (SSBs), PARP-1 hyperactivation, NAD+/ATP depletion, and µ-calpain-induced programmed necrosis. In combination with ionizing radiation (IR), beta-lapachone radiosensitized NQO1+ prostate cancer cells, under conditions where nontoxic doses of either agent alone achieved threshold levels of SSBs required for hyperactivation of PARP-1. Combination therapy significantly elevated SSBs, gamma-H2AX foci formation, and poly(ADP-ribosylation) of PARP-1, which were associated with ATP loss and induction of µ-calpain-induced programmed cell death. Radiosensitization by beta-lapachone was blocked by the NQO1 inhibitor, dicoumarol, or the PARP-1 inhibitor, DPQ. In a mouse xenograft model of prostate cancer, beta-lapachone synergized with IR to promote antitumor efficacy. NQO1 levels were elevated in ∼60% of human prostate tumors evaluated relative to adjacent normal tissue, where beta-lapachone might be efficacious alone or in combination with radiation. Our findings offer a rationale for clinical assessment of beta-lapachone (Arq501) as a radiosensitizer in prostate cancers that overexpress NQO1, offering a potentially synergistic targeting strategy to exploit PARP-1 hyperactivation. Since similar data have been found for all NQO1 endogenously over-expressing cancers, including nonsmall cell lung, pancreatic and breast cancer, this strategy should have a broad use for many cancers and in combination with many DNA damaging agents used for cancer therapy. This work was supported by NIH grant 2 R01 CA102792 to DAB Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5369. doi:10.1158/1538-7445.AM2011-5369

Abstract 2089: The role of oxidative stress in the survival of ECM-detached mammary epithelial cells

Abstract Tumorigenic mammary epithelial cells have been found to gain the ability to survive without the critical signals provided from the extracellular matrix (ECM), which are vital in normal mammary epithelial cells. However, the mechanisms underlying how cancerous epithelial cells gain the ability to survive without attachment to the ECM are poorly understood. Furthermore, the mechanisms in place to prevent survival of cells detached from the ECM are also not well characterized. Previous publications and work from our laboratory have found that there is an increase in ROS when mammary epithelial cells are detached from the ECM. In addition, it has been found that increased levels of ROS result in inhibition of fatty acid oxidation and subsequently in cell death. Neutralization of ROS in detached mammary epithelial cells through Trolox (a Vitamin E analog and antioxidant compound) treatment have been found to increase fatty acid oxidation efficacy and thereby rescue a detachment-induced loss of ATP. This suggests that detachment-induced ROS may be important in the elimination of detached cells. We are currently working on further investigating this phenomenon. Currently, we have found that other antioxidant compounds such as Vitamin C, and Epigallocatechin gallate (EGCG), also rescue the detachment-induced ATP deficiency. In addition, treating MCF-10A cells (primed for soft agar growth through the expression of HPV-E7 and Bcl-2) with these antioxidant compounds results in a significant enhancement of colony formation in soft agar. In addition to the antioxidant compound studies, we have engineered MCF-10A cells to overexpress catalase, an enzyme that efficiently neutralizes oxidative stress. We have found that directing expression of catalase to the peroxisomes results in a significant rescue of ATP levels, suggesting that the peroxisomes may be involved in this mechanism of inhibition of fatty acid oxidation. When grown in Matrigel to induce the formation of 3-dimensional structures, these newly engineered MCF-10A cells will form structures with filled lumen suggesting that catalase expression promotes the survival of ECM-detached cells. Currently, we are working to understand the source of this detachment-induced production of ROS as well as to understand the mechanism by which ROS inhibits fatty acid oxidation. Furthermore, we are working to determine the importance of neutralizing oxidative stress in tumor cell survival. In order to study the location of ROS production as well as the mechanism of fatty acid oxidation inhibition, we are utilizing siRNA/shRNA techniques, pharmacological inhibitors, and mass spectrometry. In the near future, we will use mouse xenograft studies to examine the importance of detachment-induced oxidative stress in tumor formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2089. doi:10.1158/1538-7445.AM2011-2089

Abstract 2551: The Nampt inhibitor MPC-9528 synergizes with DNA damaging agents

Abstract Background: MPC-9528 reduces cellular NAD levels and causes cell death, by blocking the NAD salvage pathway through inhibition of nicotinamide phosphoribosyltransferase (Nampt). Many DNA damaging agents also reduce cellular NAD levels, by activating the NAD consuming enzyme poly(ADP-ribose) polymerase (Parp). We hypothesized that the combination of a Nampt inhibitor and a DNA damaging agent would synergize in killing cancer cells, due to a combined effect on NAD levels through two independent mechanisms. Methods: Cellular NAD was measured using a coupled enzymatic assay. Drug combination experiments were performed in HCT116 colon carcinoma cells, using measurement of ATP levels as a cell viability endpoint. Synergy, antagonism, or additivity was assessed using the MacSynergy II program. Results: In HCT116 cells, saturating doses of MPC-9528 induced depletion of NAD with a half-life of 5 hours and a decrease in ATP that was delayed approximately 14 hours relative to NAD. Lower, sublethal concentrations of MPC-9528 induced partial NAD depletion without a concomitant ATP loss. At these sublethal concentrations, MPC-9528 was found to synergize with the DNA alkylating agents temozolomide and streptozotocin, which are known to activate Parp. Additionally, MPC-9528 was found to synergize with two structurally different thymidylate synthase inhibitors, 5-fluorouracil (5-FU) and raltitrexed, neither of which have been reported to activate Parp. Individually, 5-FU and raltitrexed each caused NAD depletion in HCT116 cells, which was enhanced by combination with MPC-9528. Furthermore, both 5-FU- and raltitrexed-mediated NAD depletion and synergy with MPC-9528 were completely blocked by the Parp inhibitor olaparib. Conclusions: Parp activation induced by the alkylating agents temozolomide and streptozotocin, or by the thymidylate synthase inhibitors 5-FU and raltitrexed, is the basis for tumoricidal synergy with the Nampt inhibitor MPC-9528. This synergy is a direct consequence of the NAD depletion resulting from Parp activation coupled with the inhibition of NAD synthesis due to Nampt inhibition. These results provide a basis for clinical combination of MPC-9528 with the agents studied here or with related agents that induce Parp activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2551. doi:10.1158/1538-7445.AM2011-2551

Endoplasmic Reticulum Stress-induced Apoptosis in Leishmania through Ca2+-dependent and Caspase-independent Mechanism

Numerous reports have shown that mitochondrial dysfunctions play a major role in apoptosis of Leishmania parasites, but the endoplasmic reticulum (ER) stress-induced apoptosis in Leishmania remains largely unknown. In this study, we investigate ER stress-induced apoptotic pathways in Leishmania major using tunicamycin as an ER stress inducer. ER stress activates the expression of ER-localized chaperone protein BIP/GRP78 (binding protein/identical to the 78-kDa glucose-regulated protein) with concomitant generation of intracellular reactive oxygen species. Upon exposure to ER stress, the elevation of cytosolic Ca(2+) level is observed due to release of Ca(2+) from internal stores. Increase in cytosolic Ca(2+) causes mitochondrial membrane potential depolarization and ATP loss as ablation of Ca(2+) by blocking voltage-gated cation channels with verapamil preserves mitochondrial membrane potential and cellular ATP content. Furthermore, ER stress-induced reactive oxygen species (ROS)-dependent release of cytochrome c and endonuclease G from mitochondria to cytosol and subsequent translocation of endonuclease G to nucleus are observed. Inhibition of caspase-like proteases with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or metacaspase inhibitor antipain does not prevent nuclear DNA fragmentation and phosphatidylserine exposure. Conversely, significant protection in tunicamycin-induced DNA degradation and phosphatidylserine exposure was achieved by either pretreatment of antioxidants (N-acetyl-L-cysteine, GSH, and L-cysteine), chemical chaperone (4-phenylbutyric acid), or addition of Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester). Taken together, these data strongly demonstrate that ER stress-induced apoptosis in L. major is dependent on ROS and Ca(2+)-induced mitochondrial toxicity but independent of caspase-like proteases.

Oxygen-Inducible Glutamate Oxaloacetate Transaminase as Protective Switch Transforming Neurotoxic Glutamate to Metabolic Fuel During Acute Ischemic Stroke

This work rests on our previous report (J Cereb Blood Flow Metab 30: 1275-1287, 2010) recognizing that glutamate (Glu) oxaloacetate transaminase (GOT) is induced when brain tissue hypoxia is corrected during acute ischemic stroke (AIS). GOT can metabolize Glu into tricarboxylic acid cycle intermediates and may therefore be useful to harness excess neurotoxic extracellular Glu during AIS as a metabolic substrate. We report that in cultured neural cells challenged with hypoglycemia, extracellular Glu can support cell survival as long as there is sufficient oxygenation. This effect is abrogated by GOT knockdown. In a rodent model of AIS, supplemental oxygen (100% O(2) inhaled) during ischemia significantly increased GOT expression and activity in the stroke-affected brain tissue and prevented loss of ATP. Biochemical analyses and in vivo magnetic resonance spectroscopy during stroke demonstrated that such elevated GOT decreased Glu levels at the stroke-affected site. In vivo lentiviral gene delivery of GOT minimized lesion volume, whereas GOT knockdown worsened stroke outcomes. Thus, brain tissue GOT emerges as a novel target in managing stroke outcomes. This work demonstrates that correction of hypoxia during AIS can help clear extracellular neurotoxic Glu by enabling utilization of this amino acid as a metabolic fuel to support survival of the hypoglycemic brain tissue. Strategies to mitigate extracellular Glu-mediated neurodegeneration via blocking receptor-mediated excitotoxicity have failed in clinical trials. We introduce the concept that under hypoglycemic conditions extracellular Glu can be transformed from a neurotoxin to a survival factor by GOT, provided there is sufficient oxygen to sustain cellular respiration.

Disruption of Pyridine Nucleotide Redox Status During Oxidative Challenge at Normal and Low-Glucose States: Implications for Cellular Adenosine Triphosphate, Mitochondrial Respiratory Activity, and Reducing Capacity in Colon Epithelial Cells

We recently demonstrated that menadione (MQ), a redox cycling quinone, mediated the loss of mitochondrial glutathione/glutathione disulfide redox balance. In this study, we showed that MQ significantly disrupted cellular pyridine nucleotide (NAD(+)/NADH, NADP(+)/NADPH) redox balance that compromised cellular ATP, mitochondrial respiratory activity, and NADPH-dependent reducing capacity in colonic epithelial cells, a scenario that was exaggerated by low glucose. In the cytosol, MQ induced NAD(+) loss concurrent with increased NADP(+) and NAD kinase activity, but decreased NADPH. In the mitochondria, NADH loss occurred in conjunction with increased nicotinamide nucleotide transhydrogenase activity and NADP(+), and decreased NADPH. These results are consistent with cytosolic NAD(+)-to-NADP(+) and mitochondrial NADH-to-NADPH shifts, but compromised NADPH availability. Thus, despite the sacrifice of NAD(+)/NADH in favor of NADPH generation, steady-state NADPH levels were not maintained during MQ challenge. Impairments of cellular bioenergetics were evidenced by ATP losses and increased mitochondrial O(2) dependence of pyridine nucleotide oxidation-reduction; half-maximal oxidation (P(50)) was 10-fold higher in low glucose, which was lowered by glutamate or succinate supplementation. This exaggerated O(2) dependence is consistent with increased O(2) diversion to nonmitochondrial O(2) consumption by MQ-semiquinone redox cycling secondary to decreased NADPH-dependent MQ detoxication at low glucose, a situation that was corrected by glucose-sparing mitochondrial substrates.

G37R SOD1 mutant alters mitochondrial complex I activity, Ca2+ uptake and ATP production

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the molecular mechanisms whereby these mutations induce motor neuron death remain controversial. Here, we show that stable overexpression of mutant human SOD1 (G37R) – but not wild-type SOD1 (wt-SOD1) – in mouse neuroblastoma cells (N2a) results in morphological abnormalities of mitochondria accompanied by several dysfunctions. Activity of the oxidative phosphorylation complex I was significantly reduced in G37R cells and correlated with lower mitochondrial membrane potential and reduced levels of cytosolic ATP. Using targeted chimeric aequorin we further analyzed the consequences of mitochondrial dysfunction on cellular Ca2+ handling. Mitochondrial Ca2+ uptake, elicited by IP3-induced Ca2+ release from endoplasmic reticulum (ER) was significantly reduced in G37R cells, while uptake induced by a brief Ca2+ pulse was not affected in permeabilized cells. The decreased mitochondrial Ca2+ uptake resulted in increased cytosolic Ca2+ transients, whereas ER Ca2+ load and resting cytosolic Ca2+ levels were not affected. Together, these findings suggest that the mechanism linking mutant G37R SOD1 and ALS involves mitochondrial respiratory chain deficiency resulting in ATP loss and impairment of mitochondrial and cytosolic Ca2+ homeostasis.

Cytotoxic effects of hydroxylated fullerenes on isolated rat hepatocytes via mitochondrial dysfunction

The cytotoxic effects of hydroxylated fullerenes, also termed fullerenols or fullerols [C(60)(OH)( n )], which are known nanomaterials and water-soluble fullerene derivatives, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C(60)(OH)(24) caused not only concentration (0-0.25mM)- and time (0-3h)-dependent cell death accompanied by the formation of cell blebs, loss of cellular ATP, reduced glutathione (GSH), and protein thiol levels, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. Of the other analogues examined, the cytotoxic effects of C(60)(OH)(12) and fullerene C(60) at a concentration of 0.125mM were less than those of C(60)(OH)(24). The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes incubated with C(60)(OH)(24) were greater than those with C(60)(OH)(12) and fullerene C(60). In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration were more markedly decreased by C(60)(OH)(24) and C(60)(OH)(12) compared with C(60). In addition, C(60)(OH)(24) and C(60)(OH)(12) resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of C(60)(OH)(12) were less than those of C(60)(OH)(24). Taken collectively, these results indicate that (a) mitochondria are target organelles for fullerenols, which elicit cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis in the early stage and subsequently oxidation of GSH and protein thiols, and lipid peroxidation through oxidative stress at a later stage; and (b) the toxic effects of fullerenols may depend on the number of hydroxyl groups participating in fullerene in rat hepatocytes.

Mechanism of ATP loss in nonoxidative contracting muscle

The transition from rest to intense exercise is a challenge to cellular energetics ([11][1], [13][2], [15][3]). The metabolic fuels, i.e., the sources of ATP to sustain muscular contraction, are creatine phosphate and glycogen. Two anaerobic metabolic paths, leading to ATP generation, are catalyzed

An Improved Isolation Procedure for Adult Mouse Cardiomyocytes

Isolated adult mouse cardiomyocytes are an important tool in cardiovascular research, but are challenging to prepare. Because the energy supply determines cell function and viability, we compared total creatine ([Cr]) and [ATP] in isolated cardiomyocytes with the intact mouse heart. Isolated myocytes suffered severe losses of Cr (-70%) and ATP (-53%). Myocytes were not able to replete [Cr] during a 5h incubation period in medium supplemented with 1mM Cr. In contrast, adding 20mM Cr to the digestion buffers was sufficient to maintain normal [Cr]. Supplementing buffers with 5mM of inosine (Ino) and adenosine (Ado) to prevent loss of cellular nucleosides partially protected against loss of ATP. To test whether maintaining [ATP] and [Cr] improves contractile function, myocytes were challenged by varying pacing rate from 0.5 to 10Hz and by adding isoproterenol (Iso) at 5 and 10Hz. All groups performed well up to 5Hz, showing a positive cell shortening-frequency relationship; however, only 16% of myocytes isolated under standard conditions were able to sustain pacing with Iso challenge at 10Hz. In contrast, 30-50% of the myocytes with normal Cr levels were able to contract and maintain low diastolic [Ca(2+)]. Cell yield also improved in Cr and the Cr/Ino/Ado-treated groups (85-90% vs. 70-75% rod shaped in untreated myocytes). These data suggest that viability and performance of isolated myocytes are improved when they are protected from the severe loss of Cr and ATP during the isolation, making them an even better research tool.

BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells

The dual functionality of the tumor suppressor BAX is implied by the nonapoptotic functions of other members of the BCL-2 family. To explore this, mitochondrial metabolism was examined in BAX-deficient HCT-116 cells as well as primary hepatocytes from BAX-deficient mice. Although mitochondrial density and mitochondrial DNA content were the same in BAX-containing and BAX-deficient cells, MitoTracker staining patterns differed, suggesting the existence of BAX-dependent functional differences in mitochondrial physiology. Oxygen consumption and cellular ATP levels were reduced in BAX-deficient cells, while glycolysis was increased. These results suggested that cells lacking BAX have a deficiency in the ability to generate ATP through cellular respiration. This conclusion was supported by detection of reduced citrate synthase activity in BAX-deficient cells. In nonapoptotic cells, a portion of BAX associated with mitochondria and a sequestered, protease-resistant form was detected. Inhibition of BAX with small interfering RNAs reduced intracellular ATP content in BAX-containing cells. Expression of either full-length or COOH-terminal-truncated BAX in BAX-deficient cells rescued ATP synthesis and oxygen consumption and reduced glycolytic activity, suggesting that this metabolic function of BAX was not dependent upon its COOH-terminal helix. Expression of BCL-2 in BAX-containing cells resulted in a subsequent loss of ATP measured, implying that, even under nonapoptotic conditions, an antagonistic interaction exists between the two proteins. These findings infer that a basal amount of BAX is necessary to maintain energy production via aerobic respiration.

Protective Effect of Liposome-Encapsulated Glutathione in a Human Epidermal Model Exposed to a Mustard Gas Analog

Sulfur mustard or mustard gas (HD) and its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES), or “half-mustard gas,” are alkylating agents that induce DNA damage, oxidative stress, and inflammation. HD/CEES are rapidly absorbed in the skin causing extensive injury. We hypothesize that antioxidant liposomes that deliver both water-soluble and lipid-soluble antioxidants protect skin cells from immediate CEES-induced damage via attenuating oxidative stress. Liposomes containing water-soluble antioxidants and/or lipid-soluble antioxidants were evaluated using in vitro model systems. Initially, we found that liposomes containing encapsulated glutathione (GSH-liposomes) increased cell viability and attenuated production of reactive oxygen species (ROS) in HaCaT cells exposed to CEES. Next, GSH-liposomes were tested in a human epidermal model, EpiDerm. In the EpiDerm, GSH-liposomes administered simultaneously or 1 hour after CEES exposure (2.5 mM) increased cell viability, inhibited CEES-induced loss of ATP and attenuated changes in cellular morphology, but did not reduce caspase-3 activity. These findings paralleled the previously described in vivo protective effect of antioxidant liposomes in the rat lung and established the effectiveness of GSH-liposomes in a human epidermal model. This study provides a rationale for use of antioxidant liposomes against HD toxicity in the skin considering further verification in animal models exposed to HD.

Functional significance of the negative-feedback regulation of ATP release via pannexin-1 hemichannels under ischemic stress in astrocytes

The opening of pannexin-1 (Px1) hemichannels is regulated by the activity of P2X7 receptors (P2X7Rs). At present, however, little is known about how extracellular ATP-sensitive P2X7Rs regulates the opening and closure of Px1 hemichannels. Several lines of evidence suggest that P2X7Rs are activated under pathological conditions such as ischemia, resulting in the opening of Px1 hemichannels responsible for the massive influx of Ca2+ from the extracellular space and the release of ATP from the cytoplasm, leading to cell death. Here we show in cultured astrocytes that the suppression of the activity of P2X7Rs during simulated ischemia (oxygen/glucose deprivation, OGD) resulted in the opening of Px1 hemichannels, leading to the enhanced release of ATP. In addition, the suppression of the activity of P2X7Rs during OGD resulted in a significant increase in astrocytic damage. Both the P2X7Rs suppression-induced enhancement of the release of ATP and cell damage were reversed by co-treatment with blockers of Px1 hemichannels, suggesting that suppression of the activity of PX7Rs resulted in the opening of Px1 hemichannels. All these findings suggested the existence of a negative-feedback loop regulating the release of ATP via Px1 hemichannels; ATP-induced suppression of ATP release. The present study indicates that ATP, released through Px1 hemichannels, activates P2X7Rs, resulting in the closure of Px1 hemichannels during ischemia. This negative-feedback mechanism, suppressing the loss of cellular ATP and Ca2+ influx, might contribute to the survival of astrocytes under ischemic stress.

Anthrolysin O and fermentation products mediate the toxicity of Bacillus anthracis to lung epithelial cells under microaerobic conditions

Bacillus anthracis generates virulence factors such as lethal and edema toxins, capsule, and hemolytic proteins under conditions of reduced oxygenation. Here, we report on the acute cytotoxicity of culture supernatants (Sups) of six nonencapsulated B. anthracis strains grown till the stationary phase under static microaerobic conditions. Human small airway epithelial, umbilical vein endothelial, Caco-2, and Hep-G2 cells were found to be susceptible. Sups displayed a reduction of pH to 5.3–5.5, indicating the onset of acid anaerobic fermentation; however, low pH itself was not a major factor of toxicity. The pore-forming hemolysin, anthrolysin O (ALO), contributed to the toxicity in a concentration-dependent manner. Its effect was found to be synergistic with a metabolic product of B. anthracis, succinic acid. Cells exposed to Sups demonstrated cytoplasmic membrane blebbing, increased permeability, loss of ATP, mitochondrial membrane potential collapse, and arrest of cell respiration. The toxicity was reduced by inhibition of ALO by cholesterol, decomposition of reactive oxygen species, and inhibition of mitochondrial succinate dehydrogenase. Cell death appears to be caused by an acute primary membrane permeabilization by ALO, followed by a burst of reactive radicals from the mitochondria fuelled by the succinate, which is generated by bacteria in the hypoxic environment. This mechanism of metabolic toxicity is relevant to the late-stage conditions of hypoxia and acidosis found in anthrax patients and might operate at anatomical locations of the host deprived from oxygen supply.

Prostate Cancer Radiosensitization through Poly(ADP-Ribose) Polymerase-1 Hyperactivation

The clinical experimental agent, β-lapachone (β-lap; Arq 501), can act as a potent radiosensitizer in vitro through an unknown mechanism. In this study, we analyzed the mechanism to determine whether β-lap may warrant clinical evaluation as a radiosensitizer. β-Lap killed prostate cancer cells by NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolic bioactivation, triggering a massive induction of reactive oxygen species, irreversible DNA single-strand breaks (SSB), poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, NAD(+)/ATP depletion, and μ-calpain-induced programmed necrosis. In combination with ionizing radiation (IR), β-lap radiosensitized NQO1(+) prostate cancer cells under conditions where nontoxic doses of either agent alone achieved threshold levels of SSBs required for hyperactivation of PARP-1. Combination therapy significantly elevated SSB level, γ-H2AX foci formation, and poly(ADP-ribosylation) of PARP-1, which were associated with ATP loss and induction of μ-calpain-induced programmed cell death. Radiosensitization by β-lap was blocked by the NQO1 inhibitor dicoumarol or the PARP-1 inhibitor DPQ. In a mouse xenograft model of prostate cancer, β-lap synergized with IR to promote antitumor efficacy. NQO1 levels were elevated in ∼60% of human prostate tumors evaluated relative to adjacent normal tissue, where β-lap might be efficacious alone or in combination with radiation. Our findings offer a rationale for the clinical utilization of β-lap (Arq 501) as a radiosensitizer in prostate cancers that overexpress NQO1, offering a potentially synergistic targeting strategy to exploit PARP-1 hyperactivation.

Doxorubicin In Vivo Rapidly Alters Expression and Translation of Myocardial Electron Transport Chain Genes, Leads to ATP Loss and Caspase 3 Activation

BackgroundDoxorubicin is one of the most effective anti-cancer drugs but its use is limited by cumulative cardiotoxicity that restricts lifetime dose. Redox damage is one of the most accepted mechanisms of toxicity, but not fully substantiated. Moreover doxorubicin is not an efficient redox cycling compound due to its low redox potential. Here we used genomic and chemical systems approaches in vivo to investigate the mechanisms of doxorubicin cardiotoxicity, and specifically test the hypothesis of redox cycling mediated cardiotoxicity.Methodology/Principal FindingsMice were treated with an acute dose of either doxorubicin (DOX) (15 mg/kg) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) (25 mg/kg). DMNQ is a more efficient redox cycling agent than DOX but unlike DOX has limited ability to inhibit gene transcription and DNA replication. This allowed specific testing of the redox hypothesis for cardiotoxicity. An acute dose was used to avoid pathophysiological effects in the genomic analysis. However similar data were obtained with a chronic model, but are not specifically presented. All data are deposited in the Gene Expression Omnibus (GEO). Pathway and biochemical analysis of cardiac global gene transcription and mRNA translation data derived at time points from 5 min after an acute exposure in vivo showed a pronounced effect on electron transport chain activity. This led to loss of ATP, increased AMPK expression, mitochondrial genome amplification and activation of caspase 3. No data gathered with either compound indicated general redox damage, though site specific redox damage in mitochondria cannot be entirely discounted.Conclusions/SignificanceThese data indicate the major mechanism of doxorubicin cardiotoxicity is via damage or inhibition of the electron transport chain and not general redox stress. There is a rapid response at transcriptional and translational level of many of the genes coding for proteins of the electron transport chain complexes. Still though ATP loss occurs with activation caspase 3 and these events probably account for the heart damage.

Mitochondrial dysfunction and biotransformation of β-carboline alkaloids, harmine and harmaline, on isolated rat hepatocytes

The cytotoxic effects and biotransformation of harmine and harmaline, which are known β-carboline alkaloids and potent hallucinogens, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to harmine caused not only concentration (0–0.50 mM)- and time (0–3 h)-dependent cell death accompanied by the formation of cell blebs and the loss of cellular ATP, reduced glutathione, and protein thiols but also the accumulation of glutathione disulfide. Of the other analogues examined, the cytotoxic effects of harmaline and harmol (a metabolite of harmine) at a concentration of 0.5 mM were less than those of harmine. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes treated with harmine were greater than those with harmaline and harmol. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration of these β-carbolines were decreased in a concentration-dependent manner. In addition, harmine resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of harmol and harmaline were less than those of harmine. At a weakly toxic level of harmine (0.25 mM), it was metabolized to harmol and its monoglucuronide and monosulfate conjugates, and the amounts of sulfate rather than glucuronide predominantly increased with time. In the presence of 2,5-dichloro-4-nitrophenol (50 μM; an inhibitor of sulfotransferase), harmine-induced cytotoxicity was enhanced, accompanied by decrease in the amount of harmol-sulfate conjugate, due to an increase in the amount of unconjugated harmol and the inhibition of harmine loss. Taken collectively, these results indicate that (a) mitochondria are target organelles for harmine, which elicits cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis; and (b) the toxic effects of harmine are greater than those of either its metabolite harmol or its analogue harmaline, suggesting that the onset of harmine-induced cytotoxicity may depend on the initial and/or residual concentrations of harmine rather than on those of its metabolites.

Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction

Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C 60OH x), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin–luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.