The HK2 Dependent "Warburg Effect" and Mitochondrial Oxidative Phosphorylation in Cancer:Targets for Effective Therapy with 3-Bromopyruvate

This review summarizes the current state of knowledge about the metabolism of cancer cells, especially with respect to the “Warburg” and “Crabtree” effects. This work also summarizes two key discoveries, one of which relates to hexokinase-2 (HK2), a major player in both the “Warburg effect” and cancer cell immortalization. The second discovery relates to the finding that cancer cells, unlike normal cells, derive as much as 60% of their ATP from glycolysis via the “Warburg effect”, and the remaining 40% is derived from mitochondrial oxidative phosphorylation. Also described are selected anticancer agents which generally act as strong energy blockers inside cancer cells. Among them, much attention has focused on 3-bromopyruvate (3BP). This small alkylating compound targets both the “Warburg effect”, i.e., elevated glycolysis even in the presence oxygen, as well as mitochondrial oxidative phosphorylation in cancer cells. Normal cells remain unharmed. 3BP rapidly kills cancer cells growing in tissue culture, eradicates tumors in animals, and prevents metastasis. In addition, properly formulated 3BP shows promise also as an effective anti-liver cancer agent in humans and is effective also toward cancers known as “multiple myeloma”. Finally, 3BP has been shown to significantly extend the life of a human patient for which no other options were available. Thus, it can be stated that 3BP is a very promising new anti-cancer agent in the process of undergoing clinical development

Secretion of Hepatitis C Virus Envelope Glycoproteins Depends on Assembly of Apolipoprotein B Positive Lipoproteins

The density of circulating hepatitis C virus (HCV) particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB) positive and triglyceride rich lipoproteins (TRL) likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP) containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1–E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed

Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes

Molecular Determinants and Dynamics of Hepatitis C Virus Secretion

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells

Association des glycoprotéines du Virus de l'Hépatite C et des apoB lipoprotéines (Vers un modèle de biosynthèse des lipo-viro-particules)

La densité des particules du virus de lhépatite C circulant dans le sang des patients infectés est très hétérogène. La densité très légère de certaines particules est liée à l'association du virus aux apoB lipoprotéines riches en triglycérides (TRL), formant une lipoprotéine hybride appelée lipo-viro-particule (LVP), contenant les glycoprotéines d'enveloppes virales E1 et E2, la capside et l'ARN viral. L'infectivité spécifique de ces particules légères est supérieure à celle des particules de plus forte densité. Le rôle des apolipoprotéines dans la synthèse et l'assemblage des particules virales est inconnu. Les cellules Caco-2 se différentient in vitro en enterocytes sécrétant de l'apoB dans des conditions particulières de culture. Après différentiation, les cellules exprimant E1 et E2 de manière stable sécrètent ces dernières de façon concomitante à la sécrétion des TRL. Elles sont détectées uniquement dans les fractions de densité contenant l'apoB et celle-ci peut être co-immunoprécipitée avec des anticorps anti-E2. Cette association avec l'apoB est aussi visualisée en microscopie électronique. La sécrétion de E1 et E2 est réduite lorsqu'on inhibe la production des TRL. E1 et E2 sont sécrétées en association avec les TRL aussi dans la lignée HepG2, mais pas dans les lignées d'hépatome Huh-7 et Huh-7.5 qui s'avèrent déficientes dans l'assemblage des lipoprotéines. E1 et E2 ont donc la capacité intrinsèque d'utiliser la machinerie d'assemblage des lipoprotéines. Un modèle d'assemblage des LVP est proposé. Les particules produites par les Caco-2 sont capables de fusionner avec des liposomes de manière dose et pH dépendante, mais pas celles produites par les Huh-7.5.The density of circulating hepatitis C virus particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles is linked to an association of the virus with apolipoprotein B (apoB) positive and triglyceride rich lipoproteins (TRL), resulting in hybrid lipoproteins known as lipo-viro-particles (LVP) containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The role of apolipoproteins in the synthesis and assembly of the viral particles is unknown? The intestinal Caco-2 cell line diferentiates in vitro into enterocyte in special conditions o culture. After one week of differentiation, Caco-2 cells stably expressing E1 and E2 secreted E1 and E2 concomitantly with TRL secretion. Secreted E1 and E2 were only detected in apoB containing density fractions. ApoB could be co-immunoprecipitated with E2-specific antibodies. ApoB and E2 association on TRL surface is also confirmed by immuno-gold labeling by using electron microscopy. E1 and E2 secretion was reduced by inhibiting the secretion of TRL. E1 and E2 were similary secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. E1 and E2 have thus the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery. A model for LVP assembly is proposed. The particles secreted by Caco-2 cells are able to fuse with liposomes in a dose and pH dependent way, whereas the particles secreted by Huh-7.5 cells are not.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Néphropathie associée au BK virus chez les transplantés rénaux (évaluation de la PCR quantitative sérique et valeur relative par rapport aux decoy cells urinaires)

LYON1-BU Santé (693882101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF