Celastrol induces unfolded protein response-dependent cell death in head and neck cancer.

The survival rate for patients with oral squamous cell carcinoma (OSCC) has not seen marked improvement in recent decades despite enhanced efforts in prevention and the introduction of novel therapies. We have reported that pharmacological exacerbation of the unfolded protein response (UPR) is an effective approach to killing OSCC cells. The UPR is executed via distinct signaling cascades whereby an initial attempt to restore folding homeostasis in the endoplasmic reticulum during stress is complemented by an apoptotic response if the defect cannot be resolved. To identify novel small molecules able to overwhelm the adaptive capacity of the UPR in OSCC cells, we engineered a complementary cell-based assay to screen a broad spectrum of chemical matter. Stably transfected CHO-K1 cells that individually report (luciferase) on the PERK/eIF2α/ATF4/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR pathways, were engineered [1]. The triterpenoids dihydrocelastrol and celastrol were identified as potent inducers of UPR signaling and cell death in a primary screen and confirmed in a panel of OSCC cells and other cancer cell lines. Biochemical and genetic assays using OSCC cells and modified murine embryonic fibroblasts demonstrated that intact PERK-eIF2-ATF4-CHOP signaling is required for pro-apoptotic UPR and OSCC death following celastrol treatment.

Large-scale analysis of UPR-mediated apoptosis in human cells.

The historic distinction between academic- and industry-driven drug discovery, whereby academicians worked to identify therapeutic targets and pharmaceutical companies advanced probe discovery, has been blurred by an academic high-throughput chemical genomic revolution. It is now common for academic labs to use biochemical or cell-based high-throughput screening (HTS) to investigate the effects of thousands or even hundreds of thousands of chemical probes on one or more targets over a period of days or weeks. To support the efforts of individual investigators, many universities have established core facilities where screening can be performed collaboratively with large chemical libraries managed by highly trained HTS personnel and guided by the experience of computational, medicinal, and synthetic organic chemists. The identification of large numbers of promising hits from such screens has driven the need for independent labs to scale down secondary in vitro assays in the hit to lead identification process. In this chapter, we will describe the use of luminescent and quantitative reverse transcription real-time PCR (qRT-PCR) technologies that permit evaluation of the expression patterns of multiple unfolded protein response (UPR) and apoptosis-related genes, and simultaneously evaluate proliferation and cell death in 96- or 384-well format.