Combined effects of GSTP1 and MRP1 in melanoma drug resistance

Glutathione-S-transferase Pi1 (GSTP1) and multidrug resistance protein 1 (MRP1) are overexpressed in melanoma, a skin cancer notoriously resistant to all current modalities of cancer therapy. To investigate the involvement of these detoxifying enzymes in the drug resistance of melanoma, an inducible (Tet-On™ system) antisense (AS) RNA strategy was used to specifically inhibit GSTP1 expression in A375 cells, a human melanoma cell line expressing high levels of GSTP1 and MRP1. Stable transfectant clones were established and analysed for GSTP1 inhibition by AS RNA. The clone A375-ASPi1, presenting a specific 40% inhibition of GSTP1 expression in the presence of doxycycline, was selected. Lowering the GSTP1 level significantly increased (about 3.3-fold) the sensitivity of A375-ASPi1 cells to etoposide. Inhibitors of glutathione synthesis (BSO), GSTs (curcumin, ethacrynic acid), and also of MRPs (MK571, sulphinpyrazone) improved the sensitising effect of GSTP1 AS RNA. All these inhibitors had stronger sensitising effects in control cells expressing high GSTP1 level (A375-ASPi1 cells in the absence of doxycycline). In conclusion, GSTP1 can act in a combined fashion with MRP1 to protect melanoma cells from toxic effects of etoposide

Biochemical warfare on the reef : the role of glutathione transferases in consumer tolerance of dietary prostaglandins

© 2010 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 5 (2010): e8537, doi:10.1371/journal.pone.0008537.Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity. The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.Financial support for this work was provided by the Ocean Life Institute Tropical Research Initiative Grant (WHOI) to KEW and MEH; the Robert H. Cole Endowed Ocean Ventures Fund (WHOI) to KEW; the National Undersea Research Center - Program Development Proposal (CMRC-03PRMN0103A) to KEW; Walter A. and Hope Noyes Smith, and a National Science Foundation Graduate Research Fellowship to KEW

The genome of the obligate intracellular parasite Trachipleistophora hominis : new insights into microsporidian genome dynamics and reductive evolution

The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome) making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome architecture in some, but not all, lineages.Publisher PDFPeer reviewe