Chemoprevention with the metabolism modifying drugs dichloroacetate and metformin in the Li-Fraumeni Syndrome model, Trp53+/- mice

BACKGROUND: While genetic testing for familial cancer has excelled, the prevention options for those carrying high risk alleles have not. Altered bioenergetics is now acknowledged as a hallmark of cancer, and several very safe drugs are available that can target this phentoype. Dichloroacetate (DCA) inactivates pyruvate dehydrogenase kinase, resulting in activation of pyruvate dehydrogenase, reduced lactic acid production and increased mitochondrial activity. Metformin, a type 2 diabetes treatment which activates AMPK, thereby inhibiting mTOR, has unambiguously been demonstrated to reduce the risk of many cancer types in diabetics. We have tested these drugs as chemopreventive agents against the mammary tumours that occur in the BALB/c-Trp53+/- mouse spontaneous tumour model. MATERIALS and METHODS Breast cancer cell lines were examined for cell viability after DCA and/or metformin treatment in vitro (neutral red uptake assay). Four groups of female BALB/c-Trp53+/- mice were given distilled water (n=75), DCA (1.5 g/L in drinking water, ~180 mg/ kg/day, n=53), metformin (0.25 g/L in drinking water, ~30 mg/kg/day, n=61) or DCA +metformin (n=51) from 8 weeks of age, and monitored for tumour development over 78 weeks, and Kaplan-Meier survival analysis was performed. RESULTS In vitro, DCA (1-5 mM) and metformin (30-300 uM), alone or combined, significantly inhibited breast cancer cell growth. In vivo, the overall tumour-free survival curves for BALB/c-Trp53+/- mice were not significantly different between treatment groups, suggesting that metformin does not reduce cancer risk in non-diabetics. However, analysis of mammary tumours alone found that DCA reduced the number and increased their latency (28.0% vs 20.8% of mice with mean latency of 55.0 vs 63.8 weeks, untreated vs DCA respectively), whereas metformin had no effect (26.2% of mice, mean latency 54.7 weeks). DCA appeared to eliminate the early onset mammary tumours (latency <52 weeks, p=0.02), while not affecting the occurrence of longer latency tumours. In contrast, the two drug combination had worse outcomes for tumour development, (35.3% of mice, latency 48.8 weeks, p<0.02 compared to DCA alone). Preliminary western blotting results in MDA-MB-468 breast cancer cells found that DCA could block the activation of AMPK by metformin, indicating the potential for drug interactions.Supported by NHMRC Career Development Award, National Breast Cancer Foundation Novel Concept Award, and Cancer Australia

Structural insights into omega-class glutathione transferases: a snapshot of enzyme reduction and identification of a non-catalytic ligandin site

Glutathione transferases (GSTs) are dimeric enzymes containing one active-site per monomer. The omega-class GSTs (hGSTO1-1 and hGSTO2-2 in humans) are homodimeric and carry out a range of reactions including the glutathione-dependant reduction of a range of compounds and the reduction of S-(phenacyl)glutathiones to acetophenones. Both types of reaction result in the formation of a mixed-disulfide of the enzyme with glutathione through the catalytic cysteine (C32). Recycling of the enzyme utilizes a second glutathione molecule and results in oxidized glutathione (GSSG) release. The crystal structure of an active-site mutant (C32A) of the hGSTO1-1 isozyme in complex with GSSG provides a snapshot of the enzyme in the process of regeneration. GSSG occupies both the G (GSH-binding) and H (hydrophobic-binding) sites and causes re-arrangement of some H-site residues. In the same structure we demonstrate the existence of a novel "ligandin" binding site deep within in the dimer interface of this enzyme, containing S-(4-nitrophenacyl)glutathione, an isozyme-specific substrate for hGSTO1-1. The ligandin site, conserved in Omega class GSTs from a range of species, is hydrophobic in nature and may represent the binding location for tocopherol esters that are uncompetitive hGSTO1-1 inhibitors.This work was supported by National Health and Medical Research Council Project Grant 366731. AJO is supported by an Australian Research Council Future Fellowship FT0990287

3D mapping of the SPRY2 domain of ryanodine receptor 1 by single-particle Cryo-EM

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca(2+) release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.The authors want to thank the Brigham and Women’s Hospital Biomedical Research Institute (to MS), the Australian National Health and the Medical Research Council (471418 to AD, MC and PB), and the European Commission (Marie Curie Action PIOF-GA-2009-237120 to AP-M)

Targeting metabolism with arsenic trioxide and dichloroacetate in breast cancer cells

Background: Cancer cells have a different metabolic profile compared to normal cells. The Warburg effect (increased aerobic glycolysis) and glutaminolysis (increased mitochondrial activity from glutamine catabolism) are well known hallmarks of cancer an

A Structural Basis for Cellular Uptake of GST-Fold Proteins

It has recently emerged that glutathione transferase enzymes (GSTs) and other structurally related molecules can be translocated from the external medium into many different cell types. In this study we aim to explore in detail, the structural features that govern cell translocation and by dissecting the human GST enzyme GSTM2-2 we quantatively demonstrate that the α-helical C-terminal domain (GST-C) is responsible for this property. Attempts to further examine the constituent helices within GST-C resulted in a reduction in cell translocation efficiency, indicating that the intrinsic GST-C domain structure is necessary for maximal cell translocation capacity. In particular, it was noted that the α-6 helix of GST-C plays a stabilising role in the fold of this domain. By destabilising the conformation of GST-C, an increase in cell translocation efficiency of up to ∼2-fold was observed. The structural stability profiles of these protein constructs have been investigated by circular dichroism and differential scanning fluorimetry measurements and found to impact upon their cell translocation efficiency. These experiments suggest that the globular, helical domain in the 'GST-fold' structural motif plays a role in influencing cellular uptake, and that changes that affect the conformational stability of GST-C can significantly influence cell translocation efficiency.This work was supported by Grant DP0558315 Australian Research Council (http://www.arc.gov.au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Do the major human glutathione S-transferases have fatty acid ethyl ester synthase activity?

AbstractTwo fatty acid ester (FAEE) synthase isoenzymes purified from human myocardium were reported to be glutathione S-transferases (GST) [(1989) Proc. Natl. Acad. Sci. USA 86, 4470–4473; and (1989) J. Clin. Invest. 84, 1942–1946]. In the present study, the FAEE synthase activity of several purified and well characterized human GSTs were examined with ethanol and [14C]oleic acid as substrates. Three isoenzymes, GST1, GST2 and GST3 which are members of the evolutionary classes μ, α and π, respectively, were studied and failed to show any significant synthesis of FAEE after 45 min incubation at 37°C, FAEE synthase activity and GST3 activity in human placental extracts can be readily separated by ion exchage chromatography on DEAE cellulose. Thus the results show that FAEE synthase activity is not a feature of the major GSTs found in human tissues. The two FAEE synthase isoenzymes isolated by Bora et al. may have been co-purified with GST isoenzymes of these FAEE synthases may be members of the GST super family that have low specific activity in conventional GST assays and have not been previously described