Combined metformin-salicylate treatment provides improved anti-tumor activity and enhanced radiotherapy response in prostate cancer; drug synergy at clinically relevant doses

BackgroundThere is need for well-tolerated therapies for prostate cancer (PrCa) secondary prevention and to improve response to radiotherapy (RT). The anti-diabetic agent metformin (MET) and the aspirin metabolite salicylate (SAL) are shown to activate AMP-activated protein kinase (AMPK), suppress de novo lipogenesis (DNL), the mammalian target of rapamycin (mTOR) pathway and reduce PrCa proliferation in-vitro. The purpose of this study was to examine whether combined MET+SAL treatment could provide enhanced PrCa tumor suppression and improve response to RT.MethodsAndrogen-sensitive (22RV1) and resistant (PC3, DU-145) PrCa cells and PC3 xenografts were used to examine whether combined treatment with MET+SAL can provide improved anti-tumor activity compared to each agent alone in non-irradiated and irradiated PrCa cells and tumors. Mechanisms of action were investigated with analysis of signaling events, mitochondria respiration and DNL activity assays.ResultsWe observed that PrCa cells are resistant to clinically relevant doses of MET. Combined MET + SAL treatment provides synergistic anti-proliferative activity at clinically relevant doses and enhances the anti-proliferative effects of RT. This was associated with suppression of oxygen consumption rate (OCR), activation of AMPK, suppression of acetyl-CoA carboxylase (ACC)-DNL and mTOR-p70^s6k/4EBP1 and HIF1α pathways. MET + SAL reduced tumor growth in non-irradiated tumors and enhanced the effects of RT.ConclusionMET+SAL treatment suppresses PrCa cell proliferation and tumor growth and enhances responses to RT at clinically relevant doses. Since MET and SAL are safe, widely-used and inexpensive agents, these data support the investigation of MET+SAL in PrCa clinical trials alone and in combination with RT.     

Molecular characterization and molasses fermentation performance of a wild yeast strain operating in an extremely wide temperature range

Molasses fermentation performance by both a cryotolerant and a thermophilic yeast (strain AXAZ-1) isolated from grapes in Greece was evaluated in an extremely wide temperature range (3–40 °C). Sequence analysis of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions assigned isolate to Saccharomyces cerevisiae. Restriction fragment length polymorphism of the mitochondrial DNA showed that strain AXAZ-1 is genetically divergent compared to other wild strains of Greek origin or commercial yeast starters. Yeast cells growing planktonically were capable of fermentation in a wide temperature spectrum, ranging from 3 °C to 38 °C. Immobilization of yeast on brewer’s spent grains (BSG) improved the thermo-tolerance of the strain and enabled fermentation at 40 °C. Time to complete fermentation with the immobilized yeast ranged from 20 days at 3 to 38 h at 40 °C. The daily ethanol productivity reached maximum (58.1 g/L) and minimum (2.5 g/L) levels at 30 and 3 °C, respectively. The aroma-related compounds’ profiles of immobilized cells at different fermentation temperatures were evaluated by using solid phase microextraction (SPME) gas chromatography–mass spectrometry (GC–MS). Molasses fermentation resulted in a high quality fermentation product due to the low concentrations of higher and amyl alcohols at all temperatures tested. Strain AXAZ-1 is very promising for the production of ethanol from low cost raw materials, as it was capable to perform fermentations of high ethanol concentration and productivities in both low and high temperatures.     

Redox Regulation of the Human Dual Specificity Phosphatase YVH1 through Disulfide Bond Formation

YVH1 was one of the first eukaryotic dual specificity phosphatases cloned, and orthologues posses a unique C-terminal zinc-coordinating domain in addition to a cysteine-based phosphatase domain. Our recent results revealed that human YVH1 (hYVH1) protects cells from oxidative stress. This function requires phosphatase activity and the zinc binding domain. This current study provides evidence that the thiol-rich zinc-coordinating domain may act as a redox sensor to impede the active site cysteine from inactivating oxidation. Furthermore, using differential thiol labeling and mass spectrometry, it was determined that hYVH1 forms intramolecular disulfide bonds at the catalytic cleft as well as within the zinc binding domain to avoid irreversible inactivation during severe oxidative stress. Importantly, zinc ejection is readily reversible and required for hYVH1 activity upon returning to favorable conditions. This inimitable mechanism provides a means for hYVH1 to remain functionally responsive for protecting cells during oxidative stimuli.Human YVH1 (hYVH1²; also known as DUSP12) is a member of the dual specificity phosphatase (DUSP) subfamily of protein-tyrosine phosphatases (PTPs) (1, 2). It is constructed of an N-terminal DUSP catalytic domain and a unique C-terminal zinc coordinating domain (3). Poor characterization and lack of mitogen-activated protein kinase targeting motifs further classify this enzyme as an atypical DUSP (1). YVH1 orthologues exhibit high evolutionary conservation and similar domain organization (3). Deletion of the yvh1 gene in yeast disrupts normal growth processes (4), whereas insertion and expression of the hyvh1 gene is capable of restoring a normal yeast growth phenotype (3). Amplification of the dusp12/hyvh1 gene has been reported in multiple sarcomas, implicating a role for hYVH1 in human disease (5–7).Recently, deletion studies from our laboratory have shown that the C-terminal zinc binding domain of hYVH1 is not essential for intrinsic phosphatase activity in vitro; however, it is required for interaction with the ATPase domain of heat shock protein 70 (8). Similarly, overexpression of wild type hYVH1 but not catalytically dead or zinc coordinating domain deletion mutants prevents cell death induced by Fas receptor activation, heat shock, and hydrogen peroxide (H₂O₂) (8). Despite these findings, current information on hYVH1 enzymatic and physiological functions remains limited.PTPs and DUSPs share similar active site architecture and catalytic mechanism, characterized by the conserved HCX₅R(S/T) motif (9, 10). The unique microenvironment within the HCX₅R(S/T) motif reduces the pK_a value of the active site cysteine, enhancing both nucleophilicity and oxidation susceptibility (11, 12). Stimulated or constituent generation of ROS can result in oxidative second messenger signaling responses capable of transient and reversible post-translational inactivation of both PTPs and DUSPs through oxidation of the catalytic cysteine (13–15).This oxidative susceptibility and modification varies among PTPs and DUSPs, a likely consequence of slight variations in active site conformations or mediated through unique regulatory domains (16–18). Accumulating evidence suggests that redox-mediated oxidation of PTPs is a dynamic modification that can differentially regulate PTPs (13, 19). Sulfenic acid, cyclic sulfenamide, and disulfide bond formation have all been shown to facilitate stable, reversible active site modifications among various PTPs and DUSPs (12, 14, 20). Furthermore, evidence suggests that oxidation predominantly and rapidly targets the active site cysteine, whereas other cysteinyl residues remain in the reduced state (15, 20).This study investigated the relationship between the zinc-coordinating C-terminal domain and the catalytic domain of hYVH1 during oxidative conditions. We provide data suggesting that the zinc binding domain can serve as a reducing agent during oxidative stress to impede the oxidation of the active site cysteine. Increased exposure to oxidative conditions readily induces disulfide bond formation within the zinc-coordinating and catalytic domains, resulting in concomitant zinc ejection and enzymatic inactivation. Zinc ejection is readily reversible and required for hYVH1 activity upon returning to reducing conditions. Thus, we propose a mechanism for phosphatase active site protection through the intrinsic redox buffering capacity of this unique zinc binding domain.     

Profound human/mouse differences in alpha-dystrobrevin isoforms: a novel syntrophin-binding site and promoter missing in mouse and rat

Background

Basoapical polarity in epithelia is critical for proper tissue function, and control of proliferation and survival. Cell culture models that recapitulate epithelial tissue architecture are invaluable to unravel developmental and disease mechanisms. Although factors important for the establishment of basal polarity have been identified, requirements for the formation of apical polarity in three-dimensional tissue structures have not been thoroughly investigated.

Results

We demonstrate that the human mammary epithelial cell line-3522 S1, provides a resilient model for studying the formation of basoapical polarity in glandular structures. Testing three-dimensional culture systems that differ in composition and origin of substrata reveals that apical polarity is more sensitive to culture conditions than basal polarity. Using a new high-throughput culture method that produces basoapical polarity in glandular structures without a gel coat, we show that basal polarity-mediated signaling and collagen IV are both necessary for the development of apical polarity.

Conclusion

These results provide new insights into the role of the basement membrane, and especially collagen IV, in the development of the apical pole, a critical element of the architecture of glandular epithelia. Also, the high-throughput culture method developed in this study should open new avenues for high-content screening of agents that act on mammary tissue homeostasis and thus, on architectural changes involved in cancer development.     

Extracting biologically significant patterns from short time series gene expression data

Background  

Time series gene expression data analysis is used widely to study the dynamics of various cell processes. Most of the time series data available today consist of few time points only, thus making the application of standard clustering techniques difficult.     

The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation

Background  

The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 ?.     

The Effect of Anisotropic Microtubule-Bound Nucleations on Ordering in the Plant Cortical Array

The highly orientationally ordered cortical microtubule array in plant cells is a key component for cell growth and development. Recent experimental and computational work has shown that the anisotropic nucleation of new microtubules from pre-existing microtubules has a major effect on the alignment process. We formulate a theoretical model to investigate the role of the microtubule-bound nucleation on the self-organization of the dynamical cortical microtubules. A bifurcation analysis of the stability of the disordered phase of the model reveals that the effective degree of co-aligned nucleation is the main determinant of the location of the transition. Increased co-aligned nucleation creates a positive feedback effect on the ordering process that can significantly widen the ordered region. We validate these predictions by comparing to the results of particle-based simulations.     

MAPKs and NF-κB differentially regulate cytokine expression in the diaphragm in response to resistive breathing: the role of oxidative stress

Inspiratory resistive breathing (IRB) induces cytokine expression in the diaphragm. The mechanism of this cytokine induction remains elusive. The roles of MAPKs and NF-κB and the impact of oxidative stress in IRB-induced cytokine upregulation in the diaphragm were studied. Wistar rats were subjected to IRB (50% of maximal inspiratory pressure) via a two-way nonrebreathing valve for 1, 3, or 6 h. Additional groups of rats subjected to IRB for 6 h were randomly assigned to receive either solvent or N-acetyl-cysteine (NAC) or inhibitors of NF-κB (BAY-11-7082), ERK1/2 (PD98059), and P38 MAPK (SB203580) to study the effect of oxidative stress, NF-κB, and MAPKs in IRB-induced cytokine upregulation in the diaphragm. Quietly breathing animals served as controls. IRB upregulated cytokine (IL-6, TNF-α, IL-10, IL-2, IL-1β) protein levels in the diaphragm and resulted in increased activation of MAPKs (P38, ERK1/2) and NF-κB. Inhibition of NF-κB and ERK1/2 blunted the upregulation of all cytokines except that of IL-6, which was further increased. P38 inhibition attenuated all cytokine (including IL-6) upregulation. Both P38 and ERK1/2 inhibition decreased NF-κB/p65 subunit phosphorylation. NAC pretreatment blunted IRB-induced cytokine upregulation in the diaphragm and resulted in decreased ERK1/2, P38, and NF-κB/p65 phosphorylation. In conclusion, IRB-induced cytokine upregulation in the diaphragm is under the regulatory control of MAPKs and NF-κB. IL-6 is regulated differently from all other cytokines through a P38-dependent and NF-κB independent pathway. Oxidative stress is a stimulus for IRB-induced cytokine upregulation in the diaphragm.     

Effects of feral cats on the evolution of anti-predator behaviours in island reptiles: insights from an ancient introduction

Exotic predators have driven the extinction of many island species. We examined impacts of feral cats on the abundance and anti-predator behaviours of Aegean wall lizards in the Cyclades (Greece), where cats were introduced thousands of years ago. We compared populations with high and low cat density on Naxos Island and populations on surrounding islets with no cats. Cats reduced wall lizard populations by half. Lizards facing greater risk from cats stayed closer to refuges, were more likely to shed their tails in a standardized assay, and fled at greater distances when approached by either a person in the field or a mounted cat decoy in the laboratory. All populations showed phenotypic plasticity in flight initiation distance, suggesting that this feature is ancient and could have helped wall lizards survive the initial introduction of cats to the region. Lizards from islets sought shelter less frequently and often initially approached the cat decoy. These differences reflect changes since islet isolation and could render islet lizards strongly susceptible to cat predation.