Sensitization of renal carcinoma cells to TRAIL-induced apoptosis by rocaglamide and analogs

Rocaglamide has been reported to sensitize several cell types to TRAIL-induced apoptosis. In recent years, advances in synthetic techniques have led to generation of novel rocaglamide analogs. However, these have not been extensively analyzed as TRAIL sensitizers, particularly in TRAIL-resistant renal cell carcinoma cells. Evaluation of rocaglamide and analogs identified 29 compounds that are able to sensitize TRAIL-resistant ACHN cells to TRAIL-induced, caspase-dependent apoptosis with sub-µM potency which correlated with their potency as protein synthesis inhibitors and with loss of cFLIP protein in the same cells. Rocaglamide alone induced cell cycle arrest, but not apoptosis. Rocaglates averaged 4–5-fold higher potency as TRAIL sensitizers than as protein synthesis inhibitors suggesting a potential window for maximizing TRAIL sensitization while minimizing effects of general protein synthesis inhibition. A wide range of other rocaglate effects (e.g. on JNK or RAF-MEK-ERK signaling, death receptor levels, ROS, ER stress, eIF4E phosphorylation) were assessed, but did not contribute to TRAIL sensitization. Other than a rapid loss of MCL-1, rocaglates had minimal effects on mitochondrial apoptotic pathway proteins. The identification of structurally diverse/mechanistically similar TRAIL sensitizing rocaglates provides insights into both rocaglate structure and function and potential further development for use in RCC-directed combination therapy.This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This research was supported [in part] by the Intramural Research Program of NIH, Frederick. National Lab, Center for Cancer Research. Research performed at Boston University was supported in part by NIH R35 GM118173. Work at the BU-CMD is supported by R24 GM111625. (HHSN261200800001E - National Cancer Institute, National Institutes of Health; Intramural Research Program of NIH, Frederick. National Lab, Center for Cancer Research; R35 GM118173 - NIH; R24 GM111625)Published versio

Erioflorin stabilizes the tumor suppressor Pdcd4 by inhibiting its interaction with the E3-ligase β-TrCP1

Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70S6K1-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition

Cultural Phylogenetics of the Tupi Language Family in Lowland South America

Background: Recent advances in automated assessment of basic vocabulary lists allow the construction of linguistic phylogenies useful for tracing dynamics of human population expansions, reconstructing ancestral cultures, and modeling transition rates of cultural traits over time. Methods: Here we investigate the Tupi expansion, a widely-dispersed language family in lowland South America, with a distance-based phylogeny based on 40-word vocabulary lists from 48 languages. We coded 11 cultural traits across the diverse Tupi family including traditional warfare patterns, post-marital residence, corporate structure, community size, paternity beliefs, sibling terminology, presence of canoes, tattooing, shamanism, men’s houses, and lip plugs. Results/Discussion: The linguistic phylogeny supports a Tupi homeland in west-central Brazil with subsequent major expansions across much of lowland South America. Consistently, ancestral reconstructions of cultural traits over the linguistic phylogeny suggest that social complexity has tended to decline through time, most notably in the independent emergence of several nomadic hunter-gatherer societies. Estimated rates of cultural change across the Tupi expansion are on the order of only a few changes per 10,000 years, in accord with previous cultural phylogenetic results in other languag

A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors

<div><p>Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein <i>in situ</i>. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format) allows quantitative analysis of protein synthesis in 384-well plates. After optimization, ICW results were compared to western blot analysis using cycloheximide as a model protein synthesis inhibitor and showed comparable results. The method was then applied to several protein synthesis inhibitors and revealed good correlation between potency as protein synthesis inhibitors to their ability to sensitize TRAIL-resistant renal carcinoma cells to TRAIL-induced apoptosis.</p></div

Effects of protein synthesis inhibitors on protein synthesis, levels of cFLIP protein, and TRAIL- induced apoptosis.

<p>(A) ACHN cells were treated for 4 h with 1 μM of the indicated compound at which point cells were processed for ICW detection and quantitation of GAPDH (open bars), puromycin (light grey), or cFLIP (dark grey). In a parallel experiment, TRAIL was added after 4 h with compounds and cell survival assessed after 24 h by the XTT assay (black bars). Signals were normalized to control on the same plate (vehicle control = 100%). Error bars represent sd (n = 4 for puromycin and GAPDH; n = 3 for cFLIP; and 3 plates, duplicate wells per plate for compounds + TRAIL). (B) Caspase 8 activity was measured after 4 h treatment with 1 μM of the indicated compound followed by 4 h in the absence (open bars) or presence (black bars) of TRAIL and normalized (vehicle control = 1.0). Error bars represent sd (n = 3). compounds: ANS: anisomycin, CHX: cycloheximide, EME: emetine, GLA: glaucarubinone, VA: verrucarin A).</p

Simultaneous visualization of puromycylated proteins (red) and GAPDH (green).

<p>(A) western blot format: ACHN cells were pretreated for 15 min ± cycloheximide (100 μM final) followed by puromycin for the indicated times. After extraction, electrophoretic separation, and transfer (25 μg/lane), detection employed anti-puromycin and anti-GAPDH followed by near IR-fluorophore-labeled secondary antibodies. (B) ICW format: ACHN cells were plated in 384-well plates at the indicated densities and treated ± puromycin (30 min) followed by analysis using the indicated antibody concentrations and visualization.</p

Quantitation of protein synthesis and inhibition by cycloheximide.

<p>(A) ICW format: ACHN cells (3000 cells/well) were treated for 15 min ± cycloheximide followed by 30 min ± puromycin. GAPDH (green) and puromycin (red) signals were visualized as described in the text. (B) Puromycin and GAPDH signals from ICW (data from panel A, represented by circles) and western blot (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165192#pone.0165192.s002" target="_blank">S2 Fig</a>—represented by squares). Cells were pretreated with 0–100 μM cycloheximide (15 min) followed by puromycin (30 min) and quantitation. In a separate ICW experiment, cells were treated with cycloheximide for 4 h then puromycin (30 min) and quantitation (represented by triangles). Black symbols represent puromycin signal, open GAPDH. All values were normalized to vehicle controls. Error bars represent sd (n = 4).</p