Franklin David Rumjanek

Haemozoin in Schistosoma mansoni.

a Departamento de Bioquimica Medica, Uni6ersidade Federal do Rio de Janeiro, Cidade Uni6ersitaria, 21941-590, Rio de Janeiro, RJ, Brazil b Di6isao de Quimica, Setor de Quimica Orgânica, CENPES, Petrobras, Cidade Uni6ersitaria, 21949-900, Rio de Janeiro, RJ, Brazil c Centro de Biociencias e Biotecnologia, Uni6ersidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, 28015-620, Brazil d Instituto de Biofisica Carlos Chagas Filho, Uni6ersidade Federal do Rio de Janeiro, Cidade Uni6ersitaria, 21941-590, Rio de Janeiro, RJ, Brazil

Inhibition of heme aggregation by chloroquine reduces Schistosoma mansoni infection

Adult Schistosoma mansoni digest large amounts of host hemoglobin and release potentially toxic heme inside their guts. We have previously demonstrated that free heme in S. mansoni is detoxified through aggregation, forming hemozoin (Hz). Possible mechanisms of heme aggregation and the effects of chloroquine (CLQ) on formation of Hz and on the viability of this parasite have now been investigated. Different fractions isolated from S. mansoni, such as crude whole-worm homogenates, total lipid extracts, and Hz itself promoted heme aggregation in vitro in a CLQ-sensitive manner. Treatment of S. mansoni-infected mice with CLQ led to remarkable decreases in total protein, Hz content, and viability of the worms, as well as in parasitemia and deposition of eggs in mouse livers. These results indicate that inhibition of formation of Hz in S. mansoni, by CLQ, led to an important decrease in the overall severity of experimental murine schistosomiasis. Taken together, the results presented here suggest that formation of Hz is a major mechanism of heme detoxification and a potential target for chemotherapy in S. mansoni.

How does the metabolism of tumour cells differ from that of normal cells

Tumour cells thrive in environments that would be hostile to their normal cell counterparts. Survival depends on the selection of cell lines that harbour modifications of both, gene regulation that shifts the balance between the cell cycle and apoptosis and those that involve the plasticity of the metabolic machinery. With regards to metabolism, the selected phenotypes usually display enhanced anaerobic glycolysis even in the presence of oxygen, the so-called Warburg effect, and anabolic pathways that provide precursors for the synthesis of lipids, proteins and DNA. The review will discuss the original ideas of Otto Warburg and how they initially led to the notion that mitochondria of tumour cells were dysfunctional. Data will be presented to show that not only the organelles are viable and respiring, but that they are key players in tumorigenesis and metastasis. Likewise, interconnecting pathways that stand out in the tumour phenotype and that require intact mitochondria such as glutaminolysis will be addressed. Furthermore, comments will be made as to how the peculiarities of the biochemistry of tumour cells renders them amenable to new forms of treatment by highlighting possible targets for inhibitors. In this respect, a case study describing the effect of a metabolite analogue, the alkylating agent 3BP (3-bromopyruvate), on glycolytic enzyme targets will be presented.

Characterisation of the structure and expression of the gene encoding a major female specific polypeptide of Schistosoma mansoni.

A previously described cDNA clone, pSF10, of Schistosoma mansoni encoding the very dominant female specific polypeptide (FSP) has been used to characterize the gene and its expression. The gene is detectable in different isolates of S. mansoni and is estimated to be present in 3 copies per haploid genome. The gene is not sex linked and exhibits neither amplification nor rearrangement concomitant with expression. Expression of the gene by parasites maturing in hamsters is first detected after 5 weeks when the RNA is present at 1/10 the level of that of 6 week worms. Although the FSP gene is specifically and highly expressed by egg laying female worms a corresponding polypeptide produced by the cell-free translation of RNA is not detectable. It was confirmed, however, that pSF10 does indeed encode a mRNA by DNA sequence analysis. The sequence demonstrated a mRNA containing a poly(A) tail and two open reading frames. One reading contains no methionine but is very high (47%) in glycine. This amino acid composition could account for the inability to detect the gene product by cell-free translation in the presence of [35S]methionine.

Methyl Jasmonate: Putative Mechanisms of Action on Cancer Cells Cycle, Metabolism, and Apoptosis

Methyl jasmonate (MJ), an oxylipid that induces defense-related mechanisms in plants, has been shown to be active against cancer cells both in vitro and in vivo, without affecting normal cells. Here we review most of the described MJ activities in an attempt to get an integrated view and better understanding of its multifaceted modes of action. MJ (1) arrests cell cycle, inhibiting cell growth and proliferation, (2) causes cell death through the intrinsic/extrinsic proapoptotic, p53-independent apoptotic, and nonapoptotic (necrosis) pathways, (3) detaches hexokinase from the voltage-dependent anion channel, dissociating glycolytic and mitochondrial functions, decreasing the mitochondrial membrane potential, favoring cytochrome c release and ATP depletion, activating pro-apoptotic, and inactivating antiapoptotic proteins, (4) induces reactive oxygen species mediated responses, (5) stimulates MAPK-stress signaling and redifferentiation in leukemia cells, (6) inhibits overexpressed proinflammatory enzymes in cancer cells such as aldo-keto reductase 1 and 5-lipoxygenase, and (7) inhibits cell migration and shows antiangiogenic and antimetastatic activities. Finally, MJ may act as a chemosensitizer to some chemotherapics helping to overcome drug resistant. The complete lack of toxicity to normal cells and the rapidity by which MJ causes damage to cancer cells turn MJ into a promising anticancer agent that can be used alone or in combination with other agents.

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.

DNA sequence profile of TP53 gene mutations in childhood B‐cell non‐Hodgkin's lymphomas: prognostic implications

Objectives: The TP53 gene encodes a nuclear protein implicated in the regulation of the cell cycle, DNA repair, and apoptosis. TP53 mutations and other alterations have been described in numerous types of tumors, and some of these have been associated with poor prognosis. The aim of this study was to characterize TP53 mutations in childhood B non‐Hodgkins lymphoma, their correlation with clinical prognostic factors and response to therapy.

Enhanced OXPHOS, glutaminolysis and β-oxidation constitute the metastatic phenotype of melanoma cells

Tumours display different cell populations with distinct metabolic phenotypes. Thus, subpopulations can adjust to different environments, particularly with regard to oxygen and nutrient availability. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focused on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated with metastasis. Melanocytes were subjected to several cycles of adhesion impairment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. Metastatic cells (4C11+) released the highest amounts of lactate, part of which was derived from glutamine catabolism. The 4C11+ cells also displayed an increased oxidative metabolism, accompanied by enhanced rates of oxygen consumption coupled to ATP synthesis. Enhanced mitochondrial function could not be explained by an increase in mitochondrial content or mitochondrial biogenesis. Furthermore, 4C11+ cells had a higher ATP content, and increased succinate oxidation (complex II activity) and fatty acid oxidation. In addition, 4C11+ cells exhibited a 2-fold increase in mitochondrial membrane potential (ΔΨmit). Consistently, functional assays showed that the migration of cells depended on glutaminase activity. Metabolomic analysis revealed that 4C11+ cells could be grouped as a subpopulation with a profile that was quite distinct from the other cells investigated in the present study. The results presented here have centred on how the multiple metabolic inputs of tumour cells may converge to compose the so-called metastatic phenotype.

Modulation of the Immune System by Ouabain

Ouabain, a known inhibitor of the Na,K‐ATPase, has been shown to regulate a number of lymphocyte functions in vitro and in vivo. Lymphocyte proliferation, apoptosis, cytokine production, and monocyte function are all affected by ouabain. The ouabain‐binding site occurs at the α subunit of the enzyme. The α subunit plays a critical role in the transport process, and four different α‐subunit isoforms have been described with different sensitivities to ouabain. Analysis by RT‐PCR indicates that α1, α2, and α3 isoforms are all present in murine lymphoid cells obtained from thymus, lymph nodes, and spleen. In these cells ouabain exerts an effect at concentrations that do not induce plasma membrane depolarization, suggesting a mechanism independent of the classical inhibition of the pump. In other systems, the Na,K‐ATPase acts as a signal transducer in addition to being an ion pump, and ouabain is capable of inducing the activation of various signal transduction cascades. Neither resting nor concanavalin A (Con A)‐activated thymocytes had their levels of phosphorylated‐extracellular signal‐regulated kinase (P‐ERK) modified by ouabain. However, ouabain decreased p38 phosphorylation induced by Con A in these cells. The pathway induced by ouabain in lymphoid cells is still unclear but might vary with the type and state of activation of the cell.

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni.

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.