HDAC inhibitors increase NRF2-signaling in tumour cells and blunt the efficacy of co-adminstered cytotoxic agents

The NRF2 signalling cascade provides a primary response against electrophilic chemicals and oxidative stress. The activation of NRF2-signaling is anticipated to have adverse clinical consequences; NRF2 is activated in a number of cancers and, additionally, its pharmacological activation by one compound can reduce the toxicity or efficiency of a second agent administered concomitantly. In this work, we have analysed systematically the ability of 152 research, pre-clinical or clinically used drugs to induce an NRF2 response using the MCF7-AREc32 NRF2 reporter. Ten percent of the tested drugs induced an NRF2 response. The NRF2 activators were not restricted to classical cytotoxic alkylating agents but also included a number of emerging anticancer drugs, including an IGF1-R inhibitor (NVP-AEW541), a PIM-1 kinase inhibitor (Pim1 inhibitor 2), a PLK1 inhibitor (BI 2536) and most strikingly seven of nine tested HDAC inhibitors. These findings were further confirmed by demonstrating NRF2-dependent induction of endogenous AKR genes, biomarkers of NRF2 activity. The ability of HDAC inhibitors to stimulate NRF2-signalling did not diminish their own potency as antitumour agents. However, when used to pre-treat cells, they did reduce the efficacy of acrolein. Taken together, our data suggest that the ability of drugs to stimulate NRF2 activity is common and should be investigated as part of the drug-development process

Application of Mice Humanised for Cytochrome P450 CYP2D6 to the Study of Tamoxifen Metabolism and Drug-Drug Interaction with Antidepressants

Tamoxifen is an estrogen receptor antagonist used in the treatment of breast cancer. It is a prodrug that is converted by several cytochrome P450 enzymes to a primary metabolite, N-desmethyltamoxifen (NDT), which is then further modified by CYP2D6 to a pharmacologically potent secondary metabolite, 4-hydroxy-N-desmethyltamoxifen (endoxifen). Antidepressants (ADs), which are often coprescribed to patients receiving tamoxifen, are also metabolized by CYP2D6 and evidence suggests that a drug–drug interaction between these agents adversely affects the outcome of tamoxifen therapy by inhibiting endoxifen formation. We evaluated this potentially important drug–drug interaction in vivo in mice humanized for CYP2D6 (hCYP2D6). The rate of conversion of NDT to endoxifen by hCYP2D6 mouse liver microsomes (MLMs) in vitro was similar to that of the most active members of a panel of 13 individual human liver microsomes. Coincubation with quinidine, a CYP2D6 inhibitor, ablated endoxifen generation by hCYP2D6 MLMs. The NDT-hydroxylation activity of wild-type MLMs was 7.4 times higher than that of hCYP2D6, whereas MLMs from Cyp2d knockout animals were inactive. Hydroxylation of NDT correlated with that of bufuralol, a CYP2D6 probe substrate, in the human liver microsome panel. In vitro, ADs of the selective serotonin reuptake inhibitor class were, by an order of magnitude, more potent inhibitors of NDT hydroxylation by hCYP2D6 MLMs than were compounds of the tricyclic class. At a clinically relevant dose, paroxetine pretreatment inhibited the generation of endoxifen from NDT in hCYP2D6 mice in vivo. These data demonstrate the potential of ADs to affect endoxifen generation and, thereby, the outcome of tamoxifen therapy

A Targeted<em> in Vivo</em> SILAC Approach for Quantification of Drug Metabolism Enzymes:Regulation by the Constitutive Androstane Receptor

The modulation of drug metabolism enzyme (DME) expression by therapeutic agents is a central mechanism of drug-drug interaction and should be assessed as early as possible in preclinical drug development. Direct measurement of DME levels is typically achieved by Western blotting, qPCR, or microarray, but these techniques have their limitations; antibody cross-reactivity among highly homologous subfamilies creates ambiguity, while discordance between mRNA and protein expression undermines observations. The aim of this study was to design a simple targeted workflow by combining in vivo SILAC and label-free proteomics approaches for quantification of DMEs in mouse liver, facilitating a rapid and comprehensive evaluation of metabolic potential at the protein level. A total of 197 peptides, representing 51 Phase I and Phase II DMEs, were quantified by LC-MS/MS using targeted high resolution single ion monitoring (tHR/SIM) with a defined mass-to-charge and retention time window for each peptide. In a constitutive androstane receptor (Car) activated mouse model, comparison of tHR/SIM-in vivo SILAC with Western blotting for analysis of the expression of cytochromes P450 was favorable, with agreement in fold-change values between methods. The tHR/SIM-in vivo SILAC approach therefore permits the robust analysis of multiple DME in a single protein sample, with clear utility for the assessment of the drug-drug interaction potential of candidate therapeutic compounds. </p

Testing the early Late Ordovician cool-water hypothesis with oxygen isotopes from conodont apatite

© 2017 Cambridge University Press. Latest Sandbian to early Katian sequences across Laurentia\u27s epicontinental sea exhibit a transition from lithologies characterized as \u27warm-water\u27 carbonates to those characterized as \u27cool-water\u27carbonates. This shift occurs across the regionally recognized M4/M5 sequence stratigraphic boundary and has been attributed to climatic cooling and glaciation, basin reorganization and upwelling of open ocean water, and/or increased water turbidity and terrigenous input associated with the Taconic tectophase. Documentation of oxygen isotopic trends across the M4/M5 and through bracketing strata provides a potential means of distinguishing among these alternative scenarios; however, oxygen isotopic records generated to date have failed to settle the debate. This lack of resolution is because δ18O records are open to multiple interpretations and potentially confounding factors related to local environmental conditions have not been tested by examining the critical interval in multiple areas and different depositional settings. To begin to address this shortcoming, we present new species-specific and mixed assemblage conodont δ18O values in samples spanning the M4/M5 boundary from the Upper Mississippi Valley, Alabama, and Virginia. The new results are combined with previous studies, providing a record of δ18O variability across SE Laurentia. The combined dataset allows us to test for regional trends at a resolution not previously available. Our results document a ~1.5‰ decrease in values across Laurentia instead of increasing δ18O values across the M4/M5 as predicted in various \u27cool-water\u27 scenarios. In short, these results do not support a shift to \u27cool-water\u27 conditions as an explanation for changes in early Katian carbonates across the M4/M5

Stimulation of paralysed quadriceps muscles with sequentially and spatially distributed electrodes during dynamic knee extension

Background: During functional electrical stimulation (FES) tasks with able-bodied (AB) participants, spatially distributed sequential stimulation (SDSS) has demonstrated substantial improvements in power output and fatigue properties compared to conventional single electrode stimulation (SES). The aim of this study was to compare the properties of SDSS and SES in participants with spinal cord injury (SCI) in a dynamic isokinetic knee extension task simulating knee movement during recumbent cycling. Method: Using a case-series design, m. vastus lateralis and medialis of four participants with motor and sensory complete SCI (AIS A) were stimulated for 6 min on both legs with both electrode setups. With SES, target muscles were stimulated by a pair of electrodes. In SDSS, the distal electrodes were replaced by four small electrodes giving the same overall stimulation frequency and having the same total surface area. Torque was measured during knee extension by a dynamometer at an angular velocity of 110 deg/s. Mean power of the left and right sides (PmeanL,R) was calculated from all stimulated extensions for initial, final and all extensions. Fatigue is presented as an index value with respect to initial power from 1 to 0, whereby 1 means no fatigue. Results: SDSS showed higher PmeanL,R values for all four participants for all extensions (increases of 132% in participant P1, 100% in P2, 36% in P3 and 18% in P4 compared to SES) and for the initial phase (increases of 84%, 59%, 66%, and 16%, respectively). Fatigue resistance was better with SDSS for P1, P2 and P4 but worse for P3 (0.47 vs 0.35, 0.63 vs 0.49, 0.90 vs 0.82 and 0.59 vs 0.77, respectively). Conclusion: Consistently higher PmeanL,R was observed for all four participants for initial and overall contractions using SDSS. This supports findings from previous studies with AB participants. Fatigue properties were better in three of the four participants. The lower fatigue resistance with SDSS in one participant may be explained by a very low muscle activation level in this case. Further investigation in a larger cohort is warranted

Collateral-resistance to estrogen and HER-activated growth is associated with modified AKT, ERα and cell-cycle signaling in a breast cancer model

These studies were supported by grants from Cancer Research UK (C503/A5010 and C1938/A6769).Aim : A model of progressively endocrine-resistant breast cancer was investigated to identify changes that can occur in signaling pathways after endocrine manipulation. Methods : The MCF7 breast cancer model is sensitive to estrogens and anti-estrogens while variant lines previously derived from wild-type MCF7 are either relatively 17β-estradiol (E2)-insensitive (LCC1) or fully resistant to estrogen and anti-estrogens (LCC9). Results : In LCC1 and LCC9 cell lines, loss of estrogen sensitivity was accompanied by loss of growth response to transforming growth factor alpha (TGFα), heregulin-beta and pertuzumab. LCC1 and LCC9 cells had enhanced AKT phosphorylation relative to MCF7 which was reflected in downstream activation of phospho-mechanistic target of rapamycin (mTOR), phospho-S6, and phospho-estrogen receptor alpha Ser167 [ERα(Ser167)]. Both AKT2 and AKT3 were phosphorylated in the resistant cell lines, but siRNA knockdown suggested that all three AKT isoforms contributed to growth response. ERα(Ser118) phosphorylation was increased by E2 and TGFα in MCF7, by E2 only in LCC1, but by neither in LCC9 cells. Multiple alterations in E2-mediated cell cycle control were identified in the endocrine-resistant cell lines including increased expression of MYC, cyclin A1, cyclin D1, cyclin-dependent kinase 1 (CDK1), CDK2, and hyperphosphorylated retinoblastoma protein (ppRb), whereas p21 and p27 were reduced. Estrogen modulated expression of these regulators in MCF7 and LCC1 cells but not in LCC9 cells. Seliciclib inhibited CDK2 activation in MCF7 cells but not in resistant variants; in all lines, it reduced ppRb, increased p53 associated responses including p21, p53 up-regulated modulator of apoptosis (PUMA), and p53 apoptosis-inducing protein 1 (p53AIP1), inhibited growth, and produced G2/M block and apoptosis. Conclusions : Multiple changes occur with progression of endocrine resistance in this model with AKT activation contributing to E2 insensitivity and loss of ERα(Ser118) phosphorylation being associated with full resistance. Cell cycle regulation is modified in endocrine-resistant breast cancer cells, and seliciclib is effective in both endocrine-sensitive and resistant diseases.Publisher PDFPeer reviewe

Stochastically-modulated inter-pulse intervals to increase the efficiency of functional electrical stimulation cycling

Introduction: Functional electrical stimulation cycling has various health benefits, but the mechanical power output and efficiency are very low compared to volitional muscle activation. Stimulation with variable frequency showed significantly higher power output values in experiments with a knee dynamometer. The aim of the present work was to compare stochastic modulation of inter-pulse interval to constant inter-pulse interval stimulation during functional electrical stimulation cycling. Methods: Seventeen able-bodied subjects participated (n = 17). Quadriceps and hamstring muscle groups were stimulated with two activation patterns: P1-constant frequency, P2-stochastic inter-pulse interval. Power output was measured on functional electrical stimulation ergometer. Results: Overall, mean power output with the stochastically modulated pattern P2 was lower than with P1 (12.57 ± 3.74 W vs. 11.44 ± 3.81 W, P1 vs. P2, p = 0.022), but no significant differences during the first 30 s and the last 30 s were observed. Conclusions: This study showed that stimulation strategies that use randomized modulation of inter-pulse intervals can negatively affect power output generation during functional electrical stimulation cycling. To minimise voluntary contractions, power measurement and assessment should be focused on the periods where only the quadriceps are stimulated.ISSN:2055-668

The Time-Domain Spectroscopic Survey: Understanding the Optically Variable Sky with SEQUELS in SDSS-III

The Time-Domain Spectroscopic Survey (TDSS) is an SDSS-IV eBOSS subproject primarily aimed at obtaining identification spectra of ~220,000 optically-variable objects systematically selected from SDSS/Pan-STARRS1 multi-epoch imaging. We present a preview of the science enabled by TDSS, based on TDSS spectra taken over ~320 deg^2 of sky as part of the SEQUELS survey in SDSS-III, which is in part a pilot survey for eBOSS in SDSS-IV. Using the 15,746 TDSS-selected single-epoch spectra of photometrically variable objects in SEQUELS, we determine the demographics of our variability-selected sample, and investigate the unique spectral characteristics inherent in samples selected by variability. We show that variability-based selection of quasars complements color-based selection by selecting additional redder quasars, and mitigates redshift biases to produce a smooth quasar redshift distribution over a wide range of redshifts. The resulting quasar sample contains systematically higher fractions of blazars and broad absorption line quasars than from color-selected samples. Similarly, we show that M-dwarfs in the TDSS-selected stellar sample have systematically higher chromospheric active fractions than the underlying M-dwarf population, based on their H-alpha emission. TDSS also contains a large number of RR Lyrae and eclipsing binary stars with main-sequence colors, including a few composite-spectrum binaries. Finally, our visual inspection of TDSS spectra uncovers a significant number of peculiar spectra, and we highlight a few cases of these interesting objects. With a factor of ~15 more spectra, the main TDSS survey in SDSS-IV will leverage the lessons learned from these early results for a variety of time-domain science applications.Comment: 17 pages, 14 figures, submitted to Ap