Determining the ability of therapeutic agents to activate the unfolded protein response in pancreatic cancer cells
The endoplasmic reticulum (ER) is one of two cellular centers of protein synthesis. When the ER is under stress, protein synthesis can be disrupted, creating misfolded proteins that build up in the ER lumen. This stress can also induce the unfolded protein response (UPR), a biological process that works to reinstate proteostasis within the ER. The UPR is activated through three transmembrane proteins: IRE1a, PERK, and ATF6; each has a unique signaling pathway to either mitigate ER stress or induce programmed death (apoptosis) of the affected cells. The apoptotic nature of the UPR gives it great potential as a target for cancer treatment. Pancreatic cancer is the third leading cause of cancer-related death in the United States, largely due to late-stage diagnosis and poor response to current treatment options. We explored the potential of UPR-augmentation, induced by various therapeutic agents, as a treatment strategy for pancreatic cancer. The therapeutic agents examined in this study were Atovaquone, GZD824, ONC201, and Triptolide, with Brefeldin A used as a positive control. Pancreatic cancer cells (MiaPaCa-2) were treated with each agent for 6 and 48 hours, then analyzed for UPR markers including phosphorylated IRE1a (pIRE1a), phosphorylated PERK (pPERK), cleaved ATF6, GRP78, and spliced XBP1. Western blotting analysis revealed varying, but increased, levels of pIRE1a, cleaved ATF6, and GRP78 in treated cells; expression levels appeared to depend on both treatment duration and agent used. Real-time PCR analysis revealed an increased level of the spliced XBP1 transcript compared to untreated samples in cell cultures treated with ONC201 (6- and 48-hour durations) and with Triptolide (6-hour duration only). These results indicate that therapeutic agents exist—so far, Triptolide and ONC201—which are capable of activating the UPR in pancreatic cancer cell lines. Further validation of these results, along with expanded investigations into optimal drug treatment duration and concentration, will be necessary.
