Complementary Cell-Based High-Throughput Screens Identify Novel Modulators of the Unfolded Protein Response

Abstract

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

Endoplasmic Reticulum Stress Sensing in the Unfolded Protein Response

“…ER stress-induced apoptosis is an important pathogenic factor in a number of widespread diseases, including diabetes, neurodegenerative diseases, atherosclerosis, and renal disease (Tabas and Ron 2011). Because of the UPR's central role in determining cell fate, there have been multiple studies to identify small molecules modulators to exploit the UPR for therapeutic benefit (Fribley et al 2011;Papandreou et al 2011;Volkmann et al 2011;Cross et al 2012;Mimura et al 2012).…”

Patulin-Induced Suicidal Erythrocyte Death

“…It contaminates further fruits, such as grapes, oranges, pears and peaches [1,3]. Cellular effects of patulin include formation of reactive oxygen species (ROS), cell cycle arrest, cytochrome c release from mitochondria, caspase-3 activation, PARP cleavage, ATF3 expression and subsequent apoptosis [1,3,4,5,6]. Patulin causes DNA damage and is mutagenic, carcinogenic and teratogenic [1,3].…”

Novel Lobophorins Inhibit Oral Cancer Cell Growth and Induce Atf4- and Chop-Dependent Cell Death in Murine Fibroblasts

“…Bioassay guided fractionation of extracts prepared from the active strain 7790_N4 allowed for the isolation of the spirotetronate macrolides lobophorin A, B, E, and F (1−4) 12,13 and three new congeners designated CR1 (5), CR2 (6), and CR3 (7) (Figure 2). Lobophorin CR1 (5) 4) showing the same shifts as 1 appeared a new quaternary carbon at δ C 71.5 instead of at δ C 52.4 or 91.0 as in lobophorin A (1) and B (2), respectively. These data indicated that instead of a nitro or amino group at position D-3, there must be a hydroxyl group, maintaining the same configuration β as in 1 and 2.…”

“…4 The UPR is a coordinated response to the accumulation of misfolded protein in the lumen of the endoplasmic reticulum (ER). Inositolrequiring enzyme 1 alpha (IRE1α), activating transcription factor 6 (ATF6), and protein kinase RNA-like endoplasmic reticulum kinase (PERK) are ER transmembrane sensors that monitor the productivity of protein folding in the lumen.…”