Microarray analysis of nemorosone-induced cytotoxic effects on pancreatic cancer cells reveals activation of the unfolded protein response (UPR)

BACKGROUND AND PURPOSE Pancreatic cancer is one of the leading cancer-related causes of death due to high chemo-resistance and fast metastasation. Nemorosone, a polycyclic polyprenylated acylphloroglucinol, has recently been identified as a promising anticancer agent. Here, we examine its growth-inhibitory effects on pancreatic cancer cells. Based on transcription profiling, a molecular mode of action is proposed. EXPERIMENTAL APPROACH Nemorosone cytotoxicity was assessed by the resazurin proliferation assay on pancreatic cancer cells and fibroblasts. Apoptosis was determined by Annexin V/propidium iodide staining as well as cytochrome c and caspase activation assays. Staining with the voltage-dependent dye JC-1 and fluorescence microscopy were used to detect effects on mitochondrial membrane potential. Total RNA was isolated from treated cell lines and subjected to microarray analysis, subsequent pathway identification and modelling. Gene expression data were validated by quantitative polymerase chain reaction and siRNA-mediated gene knock-down. KEY RESULTS Nemorosone significantly inhibited cancer cell growth, induced cytochrome c release and subsequent caspase-dependent apoptosis, rapidly abolished mitochondrial membrane potential and elevated cytosolic calcium levels, while fibroblasts were largely unaffected. Expression profiling revealed 336 genes to be affected by nemorosone. A total of 75 genes were altered in all three cell lines, many of which were within the unfolded protein response (UPR) network. DNA damage inducible transcript 3 was identified as a key regulator in UPR-mediated cell death. CONCLUSIONS AND IMPLICATIONS Nemorosone could be a lead compound for the development of novel anticancer drugs amplifying the already elevated UPR level in solid tumours, thus driving them into apoptosis. This study forms the basis for further investigations identifying nemorosone's direct molecular target(s).