Tatiana El-Bacha

Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate1

3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.

Gene expression analysis during dengue virus infection in HepG2 cells reveals virus control of innate immune response.

OBJECTIVES Liver damage occurs during Dengue Virus infection and constitutes a characteristic of severe forms of the disease. The present study was focused on the modulation of gene expression in a human hepatic cell lineage, HepG2, in response to Dengue Virus infection. METHODS The global gene expression changes in HepG2 cells after 6, 24 and 48h of infection with Dengue Virus were investigated using a new tool of microarray data analysis and real-time PCR. RESULTS HepG2 cells infected with Dengue Virus showed alterations in several signaling pathways involved in innate immune response. The analysis of pattern recognition pathways genes demonstrated that TLR3, TLR8, RIG-I and MDA5 mRNAs were up-regulated during Dengue Virus infection along with an increase in the expression of the type I interferon, IFN-beta and pro-inflammatory cytokines IL-6, IL-8 and RANTES genes. CONCLUSIONS Our results suggest that innate immune pathways are involved in the recognition of Dengue Virus by HepG2 cells. These observations may contribute to the understanding of the inflammatory responses induced by Dengue Virus-hepatocytes interaction during dengue diseases.

A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme.

Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and alpha-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32P in the gamma position ([gamma-32P]ATP), and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32P ([1-32P]fructose-1,6-bisphosphate). Activated charcoal is used to adsorb unreacted [gamma-32P]ATP, and the radioactive product in the supernatant, [1-32P]fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme.

Mayaro virus infection alters glucose metabolism in cultured cells through activation of the enzyme 6-phosphofructo 1-kinase

Although it is well established that cellular transformation with tumor virus leads to changes on glucose metabolism, the effects of cell infection by non-transforming virus are far to be completely elucidated. In this study, we report the first evidence that cultured Vero cells infected with the alphavirus Mayaro show several alterations on glucose metabolism. Infected cells presented a two fold increase on glucose consumption, accompanied by an increment in lactate production. This increase in glycolytic flux was also demonstrated by a significant increase on the activity of 6-phosphofructo 1-kinase, one of the regulatory enzymes of glycolysis. Analysis of the kinetic parameters revealed that the regulation of 6-phosphofructo 1-kinase is altered in infected cells, presenting an increase in Vmax along with a decrease in Km for fructose-6-phosphate. Another fact contributing to an increase in enzyme activity was the decrease in ATP levels observed in infected cells. Additionally, the levels of fructose 2,6-bisphosphate, a potent activator of this enzyme, was significantly reduced in infected cells. These observations suggest that the increase in PFK activity may be a compensatory cellular response to the viral-induced metabolic alterations that could lead to an impairment of the glycolytic flux and energy production. (Mol Cell Biochem 266: 191–198, 2004)

Virus-induced changes in mitochondrial bioenergetics as potential targets for therapy.

Infectious diseases such as those caused by virus, account for a vast proportion of deaths worldwide. Re-emerging aspects of host-virus interactions in recent literature include the vital role played by host metabolism on viral replication and the pro-active participation of mitochondria in this process. Different viruses use distinctive strategies to modulate mitochondrial bioenergetics and enhance viral replication. As a result, energy yielding metabolic pathways are programmed to provide both energy and biosynthetic resources to drive viral protein synthesis and produce infectious particles. Therefore, metabolic antagonists may prove important not only to outline efficient therapy strategies but also to shed light on the pathogenesis of viral infections. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Cellular distribution of phosphofructokinase activity and implications to metabolic regulation in human breast cancer

Neoplastic cells generally present profound changes in glucose metabolism. The mechanisms underlying such process are numerous and all may involve altered cellular hormonal responses. Here we report the first evidence that cellular location of phosphofructokinase activity in human breast cancer tissues is different from the one observed in control tissues and that this phenomenon may be involved in the increased glycolytic flux observed in those cells. Through co-sedimentation techniques, we observed that 60% of phosphofructokinase activity in neoplastic tissues is located in an actin-enriched fraction, against 36% in control tissues. Additionally, metastatic tumor tissues presented a two fold increase in this particulate activity when compared to non-metastatic tumor samples. We propose that the alteration in cellular distribution of phosphofructokinase activity in human breast cancer tissues is a mechanism associated to the process of cell transformation and may be a consequence of the altered hormonal milieu observed in several types of cancer.

Energy metabolism in H460 lung cancer cells: effects of histone deacetylase inhibitors.

Background Tumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls. Methodology/Principal Findings Various cellular and biochemical parameters were evaluated in lung cancer H460 cells treated with the histone deacetylase inhibitors (HDACis), sodium butyrate (NaB) and trichostatin A (TSA). NaB and TSA reduced glycolytic flux, assayed by lactate release by H460 cells in a concentration dependent manner. NaB inhibited the expression of glucose transporter type 1 (GLUT 1), but substantially increased mitochondria bound hexokinase (HK) activity. NaB induced increase in HK activity was associated to isoform HK I and was accompanied by 1.5 fold increase in HK I mRNA expression and cognate protein biosynthesis. Lactate dehydrogenase (LDH) and pyruvate kinase (PYK) activities were unchanged by HDACis suggesting that the increase in the HK activity was not coupled to glycolytic flux. High resolution respirometry of H460 cells revealed NaB-dependent increased rates of oxygen consumption coupled to ATP synthesis. Metabolomic analysis showed that NaB altered the glycolytic metabolite profile of intact H460 cells. Concomitantly we detected an activation of the pentose phosphate pathway (PPP). The high O2 consumption in NaB-treated cells was shown to be unrelated to mitochondrial biogenesis since citrate synthase (CS) activity and the amount of mitochondrial DNA remained unchanged. Conclusion NaB and TSA induced an increase in mitochondrial function and oxidative metabolism in H460 lung tumor cells concomitant with a less proliferative cellular phenotype.

Mitochondrial bioenergetic alterations in mouse neuroblastoma cells infected with Sindbis virus: implications to viral replication and neuronal death.

The metabolic resources crucial for viral replication are provided by the host. Details of the mechanisms by which viruses interact with host metabolism, altering and recruiting high free-energy molecules for their own replication, remain unknown. Sindbis virus, the prototype of and most widespread alphavirus, causes outbreaks of arthritis in humans and serves as a model for the study of the pathogenesis of neurological diseases induced by alphaviruses in mice. In this work, respirometric analysis was used to evaluate the effects of Sindbis virus infection on mitochondrial bioenergetics of a mouse neuroblastoma cell lineage, Neuro 2a. The modulation of mitochondrial functions affected cellular ATP content and this was synchronous with Sindbis virus replication cycle and cell death. At 15 h, irrespective of effects on cell viability, viral replication induced a decrease in oxygen consumption uncoupled to ATP synthesis and a 36% decrease in maximum uncoupled respiration, which led to an increase of 30% in the fraction of oxygen consumption used for ATP synthesis. Decreased proton leak associated to complex I respiration contributed to the apparent improvement of mitochondrial function. Cellular ATP content was not affected by infection. After 24 h, mitochondria dysfunction was clearly observed as maximum uncoupled respiration reduced 65%, along with a decrease in the fraction of oxygen consumption used for ATP synthesis. Suppressed respiration driven by complexes I- and II-related substrates seemed to play a role in mitochondrial dysfunction. Despite the increase in glucose uptake and glycolytic flux, these changes were followed by a 30% decrease in ATP content and neuronal death. Taken together, mitochondrial bioenergetics is modulated during Sindbis virus infection in such a way as to favor ATP synthesis required to support active viral replication. These early changes in metabolism of Neuro 2a cells may form the molecular basis of neuronal dysfunction and Sindbis virus-induced encephalitis.

1H Nuclear Magnetic Resonance Metabolomics of Plasma Unveils Liver Dysfunction in Dengue Patients

ABSTRACT Dengue, due to its global burden, is the most important arthropod-borne flavivirus disease, and early detection lowers fatality rates to below 1%. Since the metabolic resources crucial for viral replication are provided by host cells, detection of changes in the metabolic profile associated with disease pathogenesis could help with the identification of markers of prognostic and diagnostic importance. We applied 1H nuclear magnetic resonance exploratory metabolomics to study longitudinal changes in plasma metabolites in a cohort in Recife, Brazil. To gain statistical power, we used innovative paired multivariate analyses to discriminate individuals with primary and secondary infection presenting as dengue fever (DF; mild) and dengue hemorrhagic fever (DHF; severe) and subjects with a nonspecific nondengue (ND) illness (ND subjects). Our results showed that a decrease in plasma low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) discriminated dengue virus (DENV)-infected subjects from ND subjects, and also, subjects with severe infection even presented a decrease in lipoprotein concentrations compared to the concentrations in subjects with mild infection. These results add to the ongoing discussion that the manipulation of lipid metabolism is crucial for DENV replication and infection. In addition, a decrease in plasma glutamine content was characteristic of DENV infection and disease severity, and an increase in plasma acetate levels discriminated subjects with DF and DHF from ND subjects. Several other metabolites shown to be altered in DENV infection and the implications of these alterations are discussed. We hypothesize that these changes in the plasma metabolome are suggestive of liver dysfunction, could provide insights into the underlying molecular mechanisms of dengue virus pathogenesis, and could help to discriminate individuals at risk of the development of severe infection and predict disease outcome. IMPORTANCE Dengue, due to its global burden, is the most important mosquito-borne viral disease. There is no specific treatment for dengue disease, and early detection lowers fatality rates to below 1%. In this study, we observed the effects of dengue virus infection on the profile of small molecules in the blood of patients with mild and severe infection. Variations in the profiles of these small molecules reflected the replication of dengue virus in different tissues and the extent of tissue damage during infection. The results of this study showed that the molecules that changed the most were VLDL, LDL, and amino acids. We propose that these changes reflect liver dysfunction and also that they can be used to discriminate subjects with mild dengue from those with severe dengue.

Advances in the Development of Inactivated Virus Vaccines

Vaccine discovery stands out as one of the public health interventions that has achieved the greatest impact in worlds health. Vaccination is the most effective means of disease prevention, especially for viral infections. Starting with the use of smallpox vaccine by Jenner in the late 1700s, the technology for vaccine development has seen numerous advances. Currently, vaccines available for human viral illness are based on live attenuated (e.g. measles, mumps, and rubella), inactivated (e.g. hepatitis A) and recombinant (e.g. hepatitis B) viruses. Among these, inactivated vaccines are known for their easy production and safety. The present article reviews the literature and patents related to the mechanisms used for viral inactivation, mainly chemical and physical procedures, including the novel strategies that are currently being explored and that have been recently patent protected.