Adenine nucleotide translocase 2 is a key mitochondrial protein in cancer metabolism

Adenine nucleotide translocase (ANT), a mitochondrial protein that facilitates the exchange of ADP and ATP across the mitochondrial inner membrane, plays an essential role in cellular energy metabolism. Human ANT presents four isoforms (ANT1-4), each with a specific expression depending on the nature of the tissue, cell type, developmental stage and status of cell proliferation. Thus, ANT1 is specific to muscle and brain tissues; ANT2 occurs mainly in proliferative, undifferentiated cells; ANT3 is ubiquitous; and ANT4 is found in germ cells. ANT1 and ANT3 export the ATP produced by oxidative phosphorylation (OxPhos) from the mitochondria into the cytosol while importing ADP. In contrast, the expression of ANT2, which is linked to the rate of glycolytic metabolism, is an important indicator of carcinogenesis. In fact, cancers are characterized by major metabolic changes that switch cells from the normally dual oxidative and glycolytic metabolisms to an almost exclusively glycolytic metabolism. When OxPhos activity is impaired, ANT2 imports glycolytically produced ATP into the mitochondria. In the mitochondrial matrix, the F1F0-ATPase complex hydrolyzes the ATP, pumping out a proton into the intermembrane space. The reverse operations of ANT2 and F1F0-ATPase under glycolytic conditions contribute to maintaining the mitochondrial membrane potential, ensuring cell survival and proliferation. Unlike the ANT1 and ANT3 isoforms, ANT2 is not pro-apoptotic and may therefore contribute to carcinogenesis. Since the expression of ANT2 is closely linked to the mitochondrial bioenergetics of tumors, it should be taken into account for individualizing cancer treatments and for the development of anticancer strategies.     

ANT2 Isoform Required for Cancer Cell Glycolysis

The three adenine nucleotide translocator ({ANT1} to {ANT3}) isoforms, differentially expressed in human cells, play a crucial role in cell bioenergetics by catalyzing ADP and ATP exchange across the mitochondrial inner membrane. In contrast to differentiated tissue cells, transformed cells, and their ρ⁰ derivatives, i.e. cells deprived of mitochondrial DNA, sustain a high rate of glycolysis. We compared the expression pattern of {ANT} isoforms in several transformed human cell lines at different stages of the cell cycle. The level of {ANT2} expression and glycolytic ATP production in these cell lines were in keeping with their metabolic background and their state of differentiation. The sensitivity of the mitochondrial inner membrane potential (Δψ) to several inhibitors of glycolysis and oxidative phosphorylation confirmed this relationship. We propose a new model for ATP uptake in cancer cells implicating the {ANT2} isoform, in conjunction with hexokinase II and the β subunit of mitochondrial ATP synthase, in the Δψ maintenance and in the aggressiveness of cancer cells.     

Adenine nucleotide (ADP/ATP) translocase 3 participates in the tumor necrosis factor induced apoptosis of MCF-7 cells

Mitochondrial adenine nucleotide translocase (ANT) is believed to be a component or a regulatory component of the mitochondrial permeability transition pore (mtPTP), which controls mitochondrial permeability transition during apoptosis. However, the role of ANT in apoptosis is still uncertain, because hepatocytes isolated from ANT knockout and wild-type mice are equally sensitive to TNF- and Fas-induced apoptosis. In a screen for genes required for tumor necrosis factor alpha (TNF-alpha)-induced apoptosis in MCF-7 human breast cancer cells using retrovirus insertion-mediated random mutagenesis, we discovered that the ANT3 gene is involved in TNF-alpha-induced cell death in MCF-7 cells. We further found that ANT3 is selectively required for TNF- and oxidative stress-induced cell death in MCF-7 cells, but it is dispensable for cell death induced by several other inducers. This data supplements previous data obtained from ANT knockout studies, indicating that ANT is involved in some apoptotic processes. We found that the resistance to TNF-alpha-induced apoptosis observed in ANT3 mutant (ANT3(mut)) cells is associated with a deficiency in the regulation of the mitochondrial membrane potential and cytochrome c release. It is not related to intracellular ATP levels or survival pathways, supporting a previous model in which ANT regulates mtPTP. Our study provides genetic evidence supporting a role of ANT in apoptosis and suggests that the involvement of ANT in cell death is cell type- and stimulus-dependent.     

Mitochondrial UCP4 attenuates MPP+- and dopamine-induced oxidative stress,mitochondrial depolarization,and ATP deficiency in neurons and is interlinked with UCP2 expression

Mitochondrial uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis. We explored the neuroprotective role of UCP4 with its stable overexpression in SH-SY5Y cells, after exposure to either MPP⁺ or dopamine to induce ATP deficiency and oxidative stress. Cells overexpressing UCP4 proliferated faster in normal cultures and after exposure to MPP⁺ and dopamine. Differentiated UCP4-overexpressing cells survived better when exposed to MPP⁺ with decreased LDH release. Contrary to the mild uncoupling hypothesis, UCP4 overexpression resulted in increased absolute ATP levels (with ADP/ATP ratios similar to those of controls under normal conditions and ADP supplementation) associated with increased respiration rate. Under MPP⁺ toxicity, UCP4 overexpression preserved ATP levels and mitochondrial membrane potential (MMP) and reduced oxidative stress; the preserved ATP level was not due to increased glycolysis. Under MPP⁺ toxicity, the induction of UCP2 expression in vector controls was absent in UCP4-overexpressing cells, suggesting that UCP4 may compensate for UCP2 expression. UCP4 function does not seem to adhere to the mild uncoupling hypothesis in its neuroprotective mechanisms under oxidative stress and ATP deficiency. UCP4 overexpression increases cell survival by inducing oxidative phosphorylation, preserving ATP synthesis and MMP, and reducing oxidative stress.     

Oxygen consumption and expression of the adenine nucleotide translocator in cells lacking mitochondrial DNA

It has been shown previously that human rho degrees cells, deprived of mitochondrial DNA and consequently of functional oxidative phosphorylation, maintain a mitochondrial membrane potential, which is necessary for their growth. The goal of our study was to determine the precise origin of this membrane potential in three rho degrees cell lines originating from the human HepG2, 143B, and HeLa S3 cell lines. Residual cyanide-sensitive oxygen consumption suggests the persistence of residual mitochondrial respiratory chain activity, about 8% of that of the corresponding parental cells. The fluorescence emitted by the three rho degrees cell lines in the presence of a mitochondrial specific fluorochrome was partially reduced by a protonophore, suggesting the existence of a proton gradient. The mitochondrial membrane potential is maintained both by a residual proton gradient (up to 45 to 50% of the potential) and by other ion movements such as the glycolytic ATP(4-) to mitochondrial ADP(3-) exchange. The ANT2 gene, encoding isoform 2 of the adenine nucleotide translocator, is overexpressed in rho degrees HepG2 and 143B cells strongly dependent on glycolytic ATP synthesis, as compared to the corresponding parental cells, which present a more oxidative metabolism. In rho degrees HeLa S3 cells, originating from the HeLa S3 cell line, which already displays a glycolytic energy status, ANT2 gene expression was not higher as in parental cells. Mitochondrial oxygen consumption and ANT2 gene overexpression vary in opposite ways and this suggests that these two parameters have complementary roles in the maintenance of the mitochondrial membrane potential in rho degrees cells.     

Knockdown of cytosolic glutaredoxin 1 leads to loss of mitochondrial membrane potential: implication in neurodegenerative diseases

Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1), a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP), which is prevented by the thiol antioxidant, alpha-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC), an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT), an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of beta-N-oxalyl amino-L-alanine (L-BOAA), an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease), that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP.     

The Mitochondrial Unfoldase-Peptidase Complex ClpXP Controls Bioenergetics Stress and Metastasis

Mitochondria must buffer the risk of proteotoxic stress to preserve bioenergetics, but the role of these mechanisms in disease is poorly understood. Using a proteomics screen, we now show that the mitochondrial unfoldase-peptidase complex ClpXP associates with the oncoprotein survivin and the respiratory chain Complex II subunit succinate dehydrogenase B (SDHB) in mitochondria of tumor cells. Knockdown of ClpXP subunits ClpP or ClpX induces the accumulation of misfolded SDHB, impairing oxidative phosphorylation and ATP production while activating “stress” signals of 5′ adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy. Deregulated mitochondrial respiration induced by ClpXP targeting causes oxidative stress, which in turn reduces tumor cell proliferation, suppresses cell motility, and abolishes metastatic dissemination in vivo. ClpP is universally overexpressed in primary and metastatic human cancer, correlating with shortened patient survival. Therefore, tumors exploit ClpXP-directed proteostasis to maintain mitochondrial bioenergetics, buffer oxidative stress, and enable metastatic competence. This pathway may provide a “drugable” therapeutic target in cancer.     

Recovery of adriamycin induced mitochondrial dysfunction in liver by selenium

Adriamycin (ADR) is a chemotherapeutic drug. Its toxicities may associate with mitochondriopathy. Selenium (Se) is a trace element for essential intracellular antioxidant enzymes. However, there is lack of data related to the effect of selenium on the liver tissue of ADR-induced mitochondrial dysfunction. The study was to investigate whether Se could restore mitochondrial dysfunction of liver-exposed ADR. Rats were divided into four groups as a control, ADR, Se, co-treated ADR with Se groups. The biochemical measurements of the liver were made in mitochondrial and cytosol. ATP level and mitochondria membrane potential (MMP) were measured. Total oxidant (TOS), total antioxidant (TAS) status were determined and oxidative stress index (OSI) was calculated by using TOS and TAS. ADR increased TOS in mitochondria and also oxidative stress in mitochondria. ADR sligtly decreased MMP, and ATP level. Partial recovery of MMP by Se was able to elevate the ATP production in cotreatment of ADR with Se. TOS in mitochondria and cytosol was diminished, as well as OSI. We concluded that selenium could potentially be used against oxidative stress induced by ADR in liver, resulting from the restoration of MMP and ATP production and prevention of mitochondrial damage in vivo.     

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1–4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.     

Mitochondrial Function in Human Neuroblastoma Cells Is Up-Regulated and Protected by NQO1, a Plasma Membrane Redox Enzyme

Background

Recent findings suggest that NADH-dependent enzymes of the plasma membrane redox system (PMRS) play roles in the maintenance of cell bioenergetics and oxidative state. Neurons and tumor cells exhibit differential vulnerability to oxidative and metabolic stress, with important implications for the development of therapeutic interventions that promote either cell survival (neurons) or death (cancer cells).

Methods and Findings

Here we used human neuroblastoma cells with low or high levels of the PMRS enzyme NADH-quinone oxidoreductase 1 (NQO1) to investigate how the PMRS modulates mitochondrial functions and cell survival. Cells with elevated NQO1 levels exhibited higher levels of oxygen consumption and ATP production, and lower production of reactive oxygen species. Cells overexpressing NQO1 were more resistant to being damaged by the mitochondrial toxins rotenone and antimycin A, and exhibited less oxidative/nitrative damage and less apoptotic cell death. Cells with basal levels of NQO1 resulted in increased oxidative damage to proteins and cellular vulnerability to mitochondrial toxins. Thus, mitochondrial functions are enhanced and oxidative stress is reduced as a result of elevated PMRS activity, enabling cells to maintain redox homeostasis under conditions of metabolic and energetic stress.

Conclusion

These findings suggest that NQO1 is a potential target for the development of therapeutic agents for either preventing neuronal degeneration or promoting the death of neural tumor cells.     

DERA is the human deoxyribose phosphate aldolase and is involved in stress response

Deoxyribose-phosphate aldolase (EC 4.1.2.4), which converts 2-deoxy-d-ribose-5-phosphate into glyceraldehyde-3-phosphate and acetaldehyde, belongs to the core metabolism of living organisms. It was previously shown that human cells harbor deoxyribose phosphate aldolase activity but the protein responsible of this activity has never been formally identified. This study provides the first experimental evidence that DERA, which is mainly expressed in lung, liver and colon, is the human deoxyribose phosphate aldolase. Among human cell lines, the highest DERA mRNA level and deoxyribose phosphate aldolase activity were observed in liver-derived Huh-7 cells. DERA was shown to interact with the known stress granule component YBX1 and to be recruited to stress granules after oxidative or mitochondrial stress. In addition, cells in which DERA expression was down-regulated using shRNA formed fewer stress granules and were more prone to apoptosis after clotrimazole stress, suggesting the importance of DERA for stress granule formation. Furthermore, the expression of DERA was shown to permit cells in which mitochondrial ATP production was abolished to make use of extracellular deoxyinosine to maintain ATP levels. This study unraveled a previously undescribed pathway which may allow cells with high deoxyribose-phosphate aldolase activity, such as liver cells, to minimize or delay stress-induced damage by producing energy through deoxynucleoside degradation.     

Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange

Growth factor withdrawal is associated with a metabolic arrest that can result in apoptosis. Cell death is preceded by loss of outer mitochondrial membrane integrity and cytochrome c release. These mitochondrial events appear to follow a relative increase in mitochondrial membrane potential. This change in membrane potential results from the failure of the adenine nucleotide translocator (ANT)/voltage-dependent anion channel (VDAC) complex to maintain ATP/ADP exchange. Bcl-xL expression allows growth factor-deprived cells to maintain sufficient mitochondrial ATP/ADP exchange to sustain coupled respiration. These data demonstrate that mitochondrial adenylate transport is under active regulation. Efficient exchange of ADP for ATP is promoted by Bcl-xL expression permitting oxidative phosphorylation to be regulated by cellular ATP/ADP levels and allowing mitochondria to adapt to changes in metabolic demand.     

Inhibition of oxidative stress by coenzyme Q10 increases mitochondrial mass and improves bioenergetic function in optic nerve head astrocytes

Oxidative stress contributes to dysfunction of glial cells in the optic nerve head (ONH). However, the biological basis of the precise functional role of mitochondria in this dysfunction is not fully understood. Coenzyme Q10 (CoQ₁₀), an essential cofactor of the electron transport chain and a potent antioxidant, acts by scavenging reactive oxygen species (ROS) for protecting neuronal cells against oxidative stress in many neurodegenerative diseases. Here, we tested whether hydrogen peroxide (100 μM H₂O₂)-induced oxidative stress alters the mitochondrial network, oxidative phosphorylation (OXPHOS) complex (Cx) expression and bioenergetics, as well as whether CoQ₁₀ can ameliorate oxidative stress-mediated alterations in mitochondria of the ONH astrocytes in vitro. Oxidative stress triggered the activation of ONH astrocytes and the upregulation of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) protein expression in the ONH astrocytes. In contrast, CoQ₁₀ not only prevented activation of ONH astrocytes but also significantly decreased SOD2 and HO-1 protein expression in the ONH astrocytes against oxidative stress. Further, CoQ₁₀ prevented a significant loss of mitochondrial mass by increasing mitochondrial number and volume density and by preserving mitochondrial cristae structure, as well as promoted mitofilin and peroxisome-proliferator-activated receptor-γ coactivator-1 protein expression in the ONH astrocyte, suggesting an induction of mitochondrial biogenesis. Finally, oxidative stress triggered the upregulation of OXPHOS Cx protein expression, as well as reduction of cellular adeonsine triphosphate (ATP) production and increase of ROS generation in the ONH astocytes. However, CoQ₁₀ preserved OXPHOS protein expression and cellular ATP production, as well as decreased ROS generation in the ONH astrocytes. On the basis of these observations, we suggest that oxidative stress-mediated mitochondrial dysfunction or alteration may be an important pathophysiological mechanism in the dysfunction of ONH astrocytes. CoQ₁₀ may provide new therapeutic potentials and strategies for protecting ONH astrocytes against oxidative stress-mediated mitochondrial dysfunction or alteration in glaucoma and other optic neuropathies.     

Effects of formaldehyde on mitochondrial dysfunction and apoptosis in SK-N-SH neuroblastoma cells

Methanol ingestion is neurotoxic in humans due to its metabolites, formaldehyde and formic acid. Here, we compared the cytotoxicity of methanol and its metabolites on different types of cells. While methanol and formic acid did not affect the viability of the cells, formaldehyde (200–800 μg/mL) was strongly cytotoxic in all cell types tested. We investigated the effects of formaldehyde on oxidative stress, mitochondrial respiratory functions, and apoptosis on the sensitive neuronal SK-N-SH cells. Oxidative stress was induced after 2 h of formaldehyde exposure. Formaldehyde at a concentration of 400 μg/mL for 12 h of treatment greatly reduced cellular adenosine triphosphate (ATP) levels. Confocal microscopy indicated that the mitochondrial membrane potential (MMP) was dose-dependently reduced by formaldehyde. A marked and dose-dependent inhibition of mitochondrial respiratory enzymes, viz., NADH dehydrogenase (complex I), cytochrome c oxidase (complex IV), and oxidative stress-sensitive aconitase was also detected following treatment with formaldehyde. Furthermore, formaldehyde caused a concentration-dependent increase in nuclear fragmentation and in the activities of the apoptosis-initiator caspase-9 and apoptosis-effector caspase-3/-7, indicating apoptosis progression. Our data suggests that formaldehyde exerts strong cytotoxicity, at least in part, by inducing oxidative stress, mitochondrial dysfunction, and eventually apoptosis. Changes in mitochondrial respiratory function and oxidative stress by formaldehyde may therefore be critical in methanol-induced toxicity.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H(2)O(2)) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H(2)O(2)-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H(2)O(2)-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H(2)O(2), and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca(2+)) chelator, overexpression of Homer 1a had no significant effects on H(2)O(2)-induced oxidative stress. These results suggest that Homer 1a has protective effects against H(2)O(2)-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca(2+) homeostasis.