Susan P. LeDoux

Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol

BackgroundTaxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.ResultsTaxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.ConclusionLDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.

The Maintenance of Mitochondrial DNA Integrity—Critical Analysis and Update

DNA molecules in mitochondria, just like those in the nucleus of eukaryotic cells, are constantly damaged by noxious agents. Eukaryotic cells have developed efficient mechanisms to deal with this assault. The process of DNA repair in mitochondria, initially believed nonexistent, has now evolved into a mature area of research. In recent years, it has become increasingly appreciated that mitochondria possess many of the same DNA repair pathways that the nucleus does. Moreover, a unique pathway that is enabled by high redundancy of the mitochondrial DNA and allows for the disposal of damaged DNA molecules operates in this organelle. In this review, we attempt to present a unified view of our current understanding of the process of DNA repair in mitochondria with an emphasis on issues that appear controversial.

Overcoming Trastuzumab Resistance in Breast Cancer by Targeting Dysregulated Glucose Metabolism

Trastuzumab shows remarkable efficacy in treatment of ErbB2-positive breast cancers when used alone or in combination with other chemotherapeutics. However, acquired resistance develops in most treated patients, necessitating alternate treatment strategies. Increased aerobic glycolysis is a hallmark of cancer and inhibition of glycolysis may offer a promising strategy to preferentially kill cancer cells. In this study, we investigated the antitumor effects of trastuzumab in combination with glycolysis inhibitors in ErbB2-positive breast cancer. We found that trastuzumab inhibits glycolysis via downregulation of heat shock factor 1 (HSF1) and lactate dehydrogenase A (LDH-A) in ErbB2-positive cancer cells, resulting in tumor growth inhibition. Moreover, increased glycolysis via HSF1 and LDH-A contributes to trastuzumab resistance. Importantly, we found that combining trastuzumab with glycolysis inhibition synergistically inhibited trastuzumab-sensitive and -resistant breast cancers in vitro and in vivo, due to more efficient inhibition of glycolysis. Taken together, our findings show how glycolysis inhibition can dramatically enhance the therapeutic efficacy of trastuzumab in ErbB2-positive breast cancers, potentially useful as a strategy to overcome trastuzumab resistance.

Mitochondrial DNA repair in aging and disease

Mitochondria are organelles which, according to the endosymbiosis theory, evolved from purpurbacteria approximately 1.5 billion years ago. One of the unique features of mitochondria is that they have their own genome. Mitochondria replicate and transcribe their DNA semiautonomously. Like nuclear DNA, mitochondrial DNA (mtDNA) is constantly exposed to DNA damaging agents. Regarding the repair of mtDNA, the prevailing concept for many years was that mtDNA molecules suffering an excess of damage would simply be degraded to be replaced by newly generated successors copied from undamaged genomes. However, evidence now clearly shows that mitochondria contain the machinery to repair the damage to their genomes caused by certain endogenous or exogenous damaging agents. The link between mtDNA damage and repair to aging, neurodegeneration, and carcinogenesis-associated processes is the subject of this review.

Oxidative stress-induced apoptosis in neurons correlates with mitochondrial DNA base excision repair pathway imbalance

Neurodegeneration can occur as a result of endogenous oxidative stress. Primary cerebellar granule cells were used in this study to determine if mitochondrial DNA (mtDNA) repair deficiencies correlate with oxidative stress-induced apoptosis in neuronal cells. Granule cells exhibited a significantly higher intracellular oxidative state compared with primary astrocytes as well as increases in reductants, such as glutathione, and redox sensitive signaling molecules, such as AP endonuclease/redox effector factor-1. Cerebellar granule cultures also exhibited an increased susceptibility to exogenous oxidative stress. Menadione (50 μM) produced twice as many lesions in granule cell mtDNA compared with astrocytes, and granule cell mtDNA repair was significantly less efficient. A decreased capacity to repair oxidative mtDNA damage correlates strongly with mitochondrial initiated apoptosis in these neuronal cultures. Interestingly, the mitochondrial activities of initiators for base excision repair (BER), the bifunctional glycosylase/AP lyases as well as AP endonuclease, were significantly higher in cerebellar granule cells compared with astrocytes. The increased mitochondrial AP endonuclease activity in combination with decreased polymerase γ activity may cause an imbalance in oxidative BER leading to an increased production and persistence of mtDNA damage in neurons when treated with menadione. This study provides evidence linking neuronal mtDNA repair capacity with oxidative stress-related neurodegeneration.

The Amyloid β Protein Induces Oxidative Damage of Mitochondrial DNA

Multiple lines of evidence suggest involvement of oxidative stress in the pathogenesis of Alzheimer disease (AD). The finding that amyloid beta peptide (Aβ) has neurotoxic properties and that such effects are mediated in part by free-radicals has provided an avenue to explore new therapeutic strategies. In this study, we showed that exposure of PC12 cells to an Aβ fragment induces oxidative damage of mitochondrial DNA. Cells were exposed for 24 h to 50 μM Aβ (25–35) or to 50 μM of a control peptide with a scrambled sequence. Oxidative damage of mitochondrial DNA (mtDNA) was assessed using a Southern blot technique and an mtDNA-specific probe recognizing a 13.5-kilobase restriction fragment. Treatment of DNA with NaOH was used to reveal abasic sites and single strand breaks. Treatment with endonuclease II or FAPy glycosylage was used to detect pyrimidine or purine lesions, respectively. Cells exposed to Aβ exhibited marked oxidative damage of mtDNA as evidenced by characteristic changes on Southern blots. Cells exposed to the scrambled peptide did not show such modifications. Simultaneous addition of the pineal hormone melatonin consistently prevented the Aβ-induced oxidative damage to mtDNA. Mitochondrial sysfunction in AD has been demonstrated by several laboratorie. This study provides experimental evidence supporting a causative role of Aβ in mitochondrial lesions of AD.

Mechanisms of nicotinamide and thymidine protection from alloxan and streptozocin toxicity.

A common mechanism has been proposed for the β-cell toxins alloxan (ALX) and streptozocin (STZ) involving the formation of single-strand breaks in DNA that lead to the overactivation of the enzyme poly(ADP-ribose) synthetase and the critical depletion of its substrate NAD. If the toxins act via this common mechanism, the poly(ADP-ribose) synthetase inhibitors nicotinamide and thymidine would be expected to affect the formation of DNA single-strand breaks in a similar fashion. To test the effects of these inhibitors, the formation of single-strand breaks in the DNA of insulin-secreting RINr cells was monitored by assessing changes in the supercoiling of nucleoids after exposure to STZ, ALX, or methylnitrosourea (MNU). With the inclusion of nicotinamide or thymidine and STZ or MNU, more single-strand breaks in RINr cell DNA were detected. These results would be expected if nicotinamide and thymidine acted through inhibition of poly(ADP-ribose) synthetase. However, when the inhibitors were used in combination with ALX, fewer single-strand breaks were present. This suggests a reduction in ALX-induced hydroxyl radicals available to interact with DNA. Because nicotinamide has been demonstrated to be a hydroxyl-radical scavenger, the ability of thymidine to scavenge hydroxyl radicals was investigated. Thymidine, like nicotinamide, was found to be a potent scavenger of hydroxyl radicals. Thus, the mechanisms by which nicotinamide and thymidine protect against the toxic effects of STZ or ALX appear different. These findings suggest that the actions of β-cell toxins are more complex than simply the overactivation of a single enzyme.

Mechanisms of Nitrosourea-Induced β-Cell Damage: Alterations in DNA

The initial step in streptozocin (STZ)-induced β-cell toxicity has been hypothesized to be the alkylation of specific sites on DNA bases. The enzymatic removal of these lesions results in single-strand breaks that over-activate the nuclear enzyme poly(ADP-ribose) synthetase and critically deplete the cell of NAD. Our studies were performed to quantitatively evaluate the extent of DNA damage in β-cells and correlate this damage with toxicity. Monolayer cultures of neonatal rat β-cells were used to determine cytotoxicity and DNA damage after exposure to STZ or the aglycone N-methyl-N-nitrosourea (MNU). Toxicity in β-cells was determined by correlating morphological alterations observed by phase-contrast microscopy with decrements in immunoreactive insulin release. The extent of DNA damage was determined by alterations in nucleoid density and quantitation of N7-methylguanine formation. Toxicity tests revealed that STZ and MNU were not toxic at equimolar concentrations. Streptozocin was toxic at 10−3 M, whereas only mild toxicity was observed with MNU at 10−2 M. Surprisingly, however, at equimolar concentrations the two drugs caused comparable DNA-strand breaks as evidenced by their ability to shift the nucleoid migration ratio in neutral sucrose gradients. Additionally, quantitation of N7-methylguanine formation after exposure to equimolar concentrations of the drugs demonstrated that the two alkylated DNA to the same extent. These findings suggest that factors in addition to the activation of poly(ADP-ribose) synthetase must be responsible for the toxicity seen with STZ, because MNU at a nonlethal concentration is capable of causing comparable DNA damage.

Defective repair of oxidative damage in mitochondrial DNA in Down's syndrome.

Recent evidence indicates that oxidative DNA damage may be a major cause of aging. One of the more sensitive targets is the mitochondrial genome which is 10 times more susceptible to mutation than is the nuclear genome. A number of age-related neuromuscular degenerative diseases also have been associated with mutations in mitochondrial DNA (mtDNA), and progressive accumulation of oxidative damage in mtDNA from neuronal tissues over time has been shown. In support of the notion that oxidative stress leads to aging is the finding in Downs syndrome (DS), which is characterized by premature aging, that there is enhanced oxidative stress resulting from the aberrant expression of CuZn superoxide dismutase (CuZn SOD). On the basis of these observations, we hypothesized that there may be defective repair of oxidative damage in mtDNA which would ultimately lead to defective electron transport and concomitant enhanced production of reactive oxygen species (ROS). This effect would heighten the oxidative burden in the cell and accelerate the development of phenotypes associated with aging. To evaluate repair of oxidative damage in mtDNA, fibroblasts from several DS patients were treated with the reactive oxygen generator menadione. Oxidative damage was assessed at 0, 2, and 6 h after exposure using a Southern-blot technique and a mtDNA specific probe. The results of these studies show that DS cells are impaired in their ability to repair oxidative damage to mtDNA compared to age-matched control cells. Therefore, this data supports the possibility that increased production of ROS from mitochondria plays a crucial role in the development of aging phenotypes.

Alzheimer β protein mediated oxidative damage of mitochondrial DNA: Prevention by melatonin

Abstract: Most contemporary progress in Alzheimers disease (AD) stems from the study of a 42–43 amino acid peptide, called the amyloid beta protein (Aβ), as the main neuropathologic marker of the disorder. It has been demonstrated that Aβ has neurotoxic properties and that such effects are mediated by free‐radicals. Exposure of neuronal cells to Aβ results in a spectrum of oxidative lesions that are profoundly harmful to neuronal homeostasis. We had previously shown that Aβ25 35 induces oxidative damage to mitochondrial DNA (mtDNA) and that this modality of injury is prevented by melatonin. Because Aβ25‐35 does not occur in AD and because the mode of toxicity by Aβ25‐35 may be different from that of Aβ1‐42 (the physiologically relevant form of Aβ), we extended our initial observations to determine whether oxidative damage to mtDNA could also be induced by Aβ1‐42 and whether this type of injury is prevented by melatonin. Exposure of human neuroblastoma cells to Aβ1‐42 resulted in marked oxidative damage to mtDNA as determined by a quantitative polymerase chain reaction method. Addition of melatonin to cell cultures along with Aβ completely prevented the damage. This study supports previous findings with Aβ25‐35, including a causative role for Aβ in the mitochondrial oxidative lesions present in AD brains. Most important, the data confirms the neuroprotective role of melatonin in Aβ‐mediated oxidative injury. Because melatonin also inhibits amyloid aggregation, lacks toxicity, and efficiently crosses the blood‐brain barrier, this hormone appears superior to other available antioxidants as a candidate for pharmacologic intervention in AD.