Abstract
The copper(II) complex A0 induces a type of non-apoptotic cell death also known as paraptosis. Paraptosis involves extensive endoplasmic reticulum vacuolization in the absence of caspase activation. A wide panel of human cancer cell lines was used to demonstrate differences in cytotoxicity by the paraptosis-inducing drug A0 and the metal-based pro-apoptotic drug cisplatin. Gene expression profiling of the human fibrosarcoma HT1080 cells showed that, while cisplatin induced p53 targets, A0 up-regulated genes involved in the unfolded protein response (UPR) and response to heavy metals. The cytotoxic effects of A0 were associated with inhibition of the ubiquitin-proteasome system and accumulation of ubiquitinylated proteins, in a manner dependent on protein synthesis. Cycloheximide inhibited the accumulation of ubiquitinylated proteins and hampered A0-induced cell death process. The occurrence of the UPR during A0-induced death process was shown by the increased abundance of spliced XBP1 mRNA, transient eIF2alpha phosphorylation, and a series of downstream events, including attenuation of global protein synthesis and increased expression of ATF4, CHOP, BIP, and GADD34. Mouse embryonic fibroblasts expressing a mutant eIF2alpha, which could not be phosphorylated, were more resistant to A0 than wild type cells, pointing to a pro-death role of eIF2alpha phosphorylation. A0 may thus represent the prototypical member of a new class of compounds that cause paraptotic cell death via mechanisms involving eIF2alpha phosphorylation and the UPR.
Highlights
Kill cancer cells, the role of other types of cell death has been increasingly recognized in the tumor response to therapy
Differential Sensitivities of Human Cancer Cells to A0 and Cisplatin—Previous results from our laboratory have demonstrated that the copper(II) complex A0 causes non-apoptotic death of cancer cells [18]
In the present study we demonstrate that the thioxotriazole copper(II) complex A0, but not cisplatin, induces paraptoticlike cell death with the concomitant induction of the unfolded protein response (UPR)
Summary
Cell Lines and Culture—The cell human cancer cell lines Caco-2, Calu-3, HOS, HT1080, HT29, PANC-1, RD, A375, A431, A549, CAPAN-1, CFPAC-1, HeLa, HepG2, Hep, U2-OS, SAOS-2, SW872, MCF-7, MCF-7/DX, SH-SY5Y, PNT1A, PC3, 2008, C13*, and the mouse embryonal fibroblasts S/S and A/A were obtained and cultured as described under supplemental methods. At the end of the treatment cells were incubated in a resazurin solution and fluorescence recorded. Colony-forming Assay—HT1080 and HT1080PTR cells were seeded in 6-well plates at a density of 150 cells/cm and immediately treated with the indicated concentration of cisplatin. At the end of the treatments, cells and culture medium were collected, washed with PBS, and total RNA was extracted and processed as reported in supplemental methods. Culture medium was collected together with the cells and samples processed as described under supplemental methods. Cells were incubated with 488Alexafluor goat anti-rabbit antibody (1:800) and visualized by means of Nikon Eclipse 300 inverted fluorescence microscope. At the end of the treatment the culture medium was collected from each well and briefly centrifuged to pellet floating cells and debris. Fluorescent GFP- or PI-positive cells were counted on the digital images
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
