Javier Márquez

Intracellular redox status and oxidative stress: implications for cell proliferation, apoptosis, and carcinogenesis.

Oxidative stress can be defined as the imbalance between cellular oxidant species production and antioxidant capability. Reactive oxygen species (ROS) are involved in a variety of different cellular processes ranging from apoptosis and necrosis to cell proliferation and carcinogenesis. In fact, molecular events, such as induction of cell proliferation, decreased apoptosis, and oxidative DNA damage have been proposed to be critically involved in carcinogenesis. Carcinogenicity and aging are characterized by a set of complex endpoints, which appear as a series of molecular reactions. ROS can modify many intracellular signaling pathways including protein phosphatases, protein kinases, and transcription factors, suggesting that the majority of the effects of ROS are through their actions on signaling pathways rather than via non-specific damage of macromolecules; however, exact mechanisms by which redox status induces cells to proliferate or to die, and how oxidative stress can lead to processes evoking tumor formation are still under investigation.

Glutamine and its relationship with intracellular redox status, oxidative stress and cell proliferation/death

Glutamine is a multifaceted amino acid used for hepatic urea synthesis, renal ammoniagenesis, gluconeogenesis in both liver and kidney, and as a major respiratory fuel for many cells. Decreased glutamine concentrations are found during catabolic stress and are related to susceptibility to infections. Besides, glutamine is not only an important energy source in mitochondria, but is also a precursor of the brain neurotransmitter glutamate, which is likewise used for biosynthesis of the cellular antioxidant glutathione. Reactive oxygen species, such as superoxide anions and hydrogen peroxide, function as intracellular second messengers activating, among others, apoptosis, whereas glutamine is an apoptosis suppressor. In fact, it could contribute to block apoptosis induced by exogenous agents or by intracellular stimuli. In conclusion, this article shows evidences for the important role of glutamine in the regulation of the cellular redox balance, including brain oxidative metabolism, apoptosis and tumour cell proliferation.

Oxidative stress in apoptosis and cancer: an update

The oxygen paradox tells us that oxygen is both necessary for aerobic life and toxic to all life forms. Reactive oxygen species (ROS) touch every biological and medical discipline, especially those involving proliferative status, supporting the idea that active oxygen may be increased in tumor cells. In fact, metabolism of oxygen and the resulting toxic byproducts can cause cancer and death. Efforts to counteract the damage caused by ROS are gaining acceptance as a basis for novel therapeutic approaches, and the field of prevention of cancer is experiencing an upsurge of interest in medically useful antioxidants. Apoptosis is an important means of regulating cell numbers in the developing cell system, but it is so important that it must be controlled. Normal cell death in homeostasis of multicellular organisms is mediated through tightly regulated apoptotic pathways that involve oxidative stress regulation. Defective signaling through these pathways can contribute to both unbalance in apoptosis and development of cancer. Finally, in this review, we discuss new knowledge about recent tools that provide powerful antioxidant strategies, and designing methods to deliver to target cells, in the prevention and treatment of cancer.

Relevance of glutamine metabolism to tumor cell growth

Tumor cells are characterized as rapidly dividing cells, and consequently they need a constant supply of both energy and nitrogen substrates. To resolve their energy requirements, they are able to use virtually any substrate: glucose [see 1 for a review; 2-4], lipids [5-7], ketone bodies [3], even amino acids [2-4, 8-10]. Nevertheless, the glucose and amino acid consumption by malignant tumor cells overcomes their own needs for their metabolic requirements; thus, tumor cells apparently waste glucose and amino acids without any profit [1, ll]. In this context, tumor has been described as a trap for glucose and nitrogen [12-13]. Tumors compete with the host for glucose [13-14]; this competence results in a progressive hypoglycemia [15] and host hepatic glycogen depletion [13]. In the same way, tumors compete for nitrogen compounds; this process produces in the host a negative nitrogen balance and a characteristic weight loss, and in the tumor a reciprocal nitrogen increase. The biochemical mechanisms underlying these phenomena still remain unclear. There is consensus that tumors increase protein degradation and reduce protein synthesis in the host tissues [16]. Alanine and glutamine are two efficient vehicles for the transport of nitrogen and carbon-skeletons between the different tissues in the living organism [17-18]. When a tumor develops, there is a net flux of amino acids from host tissues to the tumor [19]. Since ammonium ions are very toxic for most of the cells, glutamine is the physiological non-toxic ammonium vehicle between different mammalian tissues; therefore, glutamine is the main source of nitrogen for tumor cells [2, 20-21]. Thus, the presence of a tumor must produce great changes in the metabolism of glutamine in host tissues in such a way that host nitrogen metabolism is accomodated to tumor enhanced requirements of glutamine. To be used, glutamine must be transported into tumor mitochondria, where it is metabolized [21]. This implies two transport processes: the transport of glutamine across the plasma membrane and across the inner mitochondrial membrane. Once glutamine has been incorporated into tumor cells, this amino acid is quickly metabolized [12, 16, 19].

Roles of dioxins and heavy metals in cancer and neurological diseases using ROS-mediated mechanisms

Oxidants have critical functions inside healthy and unhealthy cells. Deregulated cell cycle and apoptosis, both regulated by oxidative stress, have been described as hallmarks of mitotic (cancer) and postmitotic (neuronal) cells. This review provides an updated revision of the oxidant effects of some environmental contaminants such as dioxins and the heavy metals cadmium, cobalt, and copper. Dioxins exert their toxic actions by acting on phase I and phase II enzymes, such as cytochromes P450, superoxide dismutase, and glutathione peroxidase, promoting cell proliferation, growth arrest, and apoptosis, affecting cancer homeostasis and neuronal function. Heavy metals manifest cytotoxic effects in various cells and tissues, and tight regulation of metals is essential to the health of organisms. Cadmium modulates gene expression and signal transduction and reduces activities of proteins involved in antioxidant defense, interfering with DNA repair and modifying cancer development and brain function. Cobalt provokes generation of reactive oxygen species and DNA damage in cancer cells and brain tissues, altering proliferation and differentiation and causing apoptosis. Copper is a key metal in cell division processes in both normal and tumor cells. Copper also has been shown to have an important role in neurodegenerative diseases such as Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis.

Identification of two human glutaminase loci and tissue-specific expression of the two related genes.

Abstract. Glutaminolysis is initiated by either of two isoforms, K- and L-types, of the enzyme phosphate-activated glutaminase. The chromosomal localization, genomic organization, and the tissue-specific expression of the genes have been investigated in the human by using isoform-specific cDNA probes. Results obtained from radiation hybrid mapping experiments assigned the K-glutaminase gene to human Chromosome (Chr) 2, and a second locus for l-glutaminase in Chr 12 was identified. Southern blot analysis with the L-cDNA probe showed hybridization to a single restriction fragment, while four to seven fragments were found to hybridize to the K-cDNA probe. The distribution of human glutaminase expression was also investigated: the L-cDNA probe detected a single band of 2.4 kb in liver, brain, and pancreas, whereas a single transcript of approximately 4.4 kb was detected in kidney, brain, heart, placenta, lung, and pancreas by using the K-cDNA probe. This work provides evidence that the human liver and kidney glutaminase isozymes are encoded by separate genes located on different chromosomes; furthermore, the expression pattern in human tissues revealed for both isoenzymes differs notably from the paradigm based upon the isoenzyme distribution in rats.

Glutamine homeostasis and mitochondrial dynamics

Glutamine is a multifaceted amino acid that plays key roles in many metabolic pathways and also fulfils essential signaling functions. Although classified as non-essential, recent evidence suggests that glutamine is a conditionally essential amino acid in several physiological situations. Glutamine homeostasis must therefore be exquisitely regulated and mitochondria represent a major site of glutamine metabolism in numerous cell types. Glutaminolysis is mostly a mitochondrial process with repercussions in organelle structure and dynamics suggesting a tight and mutual control between mitochondrial form and cell bioenergetics. In this review we describe an updated account focused on the critical involvement of glutamine in oxidative stress, mitochondrial dysfunction and tumour cell proliferation, with special emphasis in the initial steps of mitochondrial glutamine pathways: transport into the organelle and hydrolytic deamidation through glutaminase enzymes. Some controversial issues about glutamine catabolism within mitochondria are also reviewed.

Ehrlich ascites tumour unbalances splenic cell populations and reduces responsiveness of T cells to Staphylococcus aureus enterotoxin B stimulation

Tumours must avoid host immune response to survive and proliferate; to achieve this purpose, tumours interact with cells of the immune system by means of tumour secreted factors. The alterations of splenic cell populations in mice bearing the Ehrlich ascites tumour have been studied. A rapid and acute response was observed, characterized by a decrease in both CD4 and CD8 T cells, and a transient increase in the number of B cells, which peaked 2 days after tumour inoculation. An increase in macrophage population and in the homing antigen CD18 was also detected. In vitro incubations of splenic cells with the Staphylococcus aureus enterotoxin B (SEB) showed that tumour induces a state of reduced responsiveness to stimulation of T cells, mainly affecting CD8 T cells, and a diminished IFN-gamma expression.

Co-expression of glutaminase K and L isoenzymes in human tumour cells

The pattern of expression of glutaminase isoenzymes in tumour cells has been investigated to clarify its role in the malignant transformation and the prospect of its use as a clinically relevant factor. Using leukaemia cells from medullar blood of human patients and several established human cancer cell lines, we have developed a competitive RT (reverse transcriptase)-PCR assay to quantify simultaneously K-type (kidney-type) and L-type (liver-type) glutaminase mRNAs. Co-expression of both transcripts and higher amounts of L-type mRNA were always found in all cancer cell types analysed. However, mature lymphocytes from the medullar blood of a patient suffering aplasia did not express the K-type transcript and showed a 15-fold increase of L-type transcript. Co-expression was also confirmed at the protein level using isoform-specific antibodies; nevertheless, it did not correlate with the relative abundance of glutaminase transcripts and strong K-type protein signals were detected. On the other hand, marked differences were found with regard to glutamate inhibition and phosphate activation of tumour glutaminase activity. Taken together, the protein data suggest that K isoform would account for the majority of glutaminase activity in these human tumour cells. The results confirm that simultaneous expression of both isoenzymes in human cancer cells is a more frequent event than previously thought. Furthermore, the present work and other previous data suggest that K isoform is up-regulated with increased rates of proliferation, whereas prevalence of the L isoform seems to be related with resting or quiescent cell states.

Glutaminase isoenzymes as key regulators in metabolic and oxidative stress against cancer.

Cancer cells require a robust supply of reduced nitrogen to produce nucleotides, non-essential amino acids and a high cellular redox activity. Glutamine provides a major substrate for respiration as well as nitrogen for the production of proteins, hexosamines, and macromolecules. Therefore, glutamine is one of key molecules in cancer metabolism during cell proliferation. The notion of targeting glutamine metabolism in cancer, originally rationalized by the number of pathways fed by this nutrient, has been reinforced by more recent studies demonstrating that its metabolism is regulated by oncogenes. Glutamine can exert its effects by modulating redox homeostasis, bioenergetics, nitrogen balance or other functions, including by being a precursor of glutathione, the major nonenzymatic cellular antioxidant. Glutaminase (GA) is the first enzyme that converts glutamine to glutamate, which is in turn converted to alpha-ketoglutarate for further metabolism in the tricarboxylic acid cycle. Different GA isoforms in mammals are encoded by two genes, Gls and Gls2. As each enzymatic form of GA has distinct kinetic and molecular characteristics, it has been speculated that the differential regulation of GA isoforms may reflect distinct functions or requirements in different tissues or cell states. GA encoded by Gls gene (GLS) has been demonstrated to be regulated by oncogenes and to support tumor cell growth. GA encoded by Gls2 gene (GLS2) reduces cellular sensitivity to reactive oxygen species associated apoptosis possibly through glutathione-dependent antioxidant defense, and therefore to behave more like a tumor suppressor. Thus, modulation of GA function may be a new therapeutic target for cancer treatment.