Paraptotic Cell Death Induced by the Thioxotriazole Copper Complex A0: A New Tool to Kill Apoptosis-Resistant Cancer Cells

Abstract

The copper(II) complex A0 induces non-apoptotic programmed cell death in human HT1080 fibrosarcoma cells but not in normal fibroblasts (J Med Chem, 50(8):1916–1924, 2007). While typical apoptotic features, such as caspase-3 activation or nuclear fragmentation, are evident in cisplatin-treated cells, they are absent in A0-dependent cell death. In contrast, the latter process is hallmarked by the development of huge vacuoles originating from endoplasmic reticulum (Histochem Cell Biol 126(4):473–482, 2006), a feature consistent with the newly described type of cell death named paraptosis (PNAS 97(26):14376–14381, 2000). Consistently, in a panel of human cancer cells there is no correlation between the sensitivities to A0 and cis-platin. In the same panel, paraptosis-like cell death is observed in all the A0-sensitive cell lines. Moreover, the copper complex kills cisplatin-sensitive cells (HT1080 and ovarian carcinoma 2008) as well as their cisplatin-resistant counterparts (C13* cells and the newly established HT1080PTR line) with comparable potencies. The different activity spectrum between A0 and cisplatin suggests distinct mechanisms of action for the two drugs. In agreement with this hypothesis, a whole-genome expression analysis, performed in HT1080 cells, showed that the transcriptional response evoked by the two drugs is poorly overlapping. A0 induces genes involved in oxidative- and endoplasmic reticulum-stress (ER stress), while cisplatin increases the expression of typical p53 targets. Moreover, A0 strongly induces metal responsive genes, as well as HSPs, chaperones and other genes involved in the Unfolded Protein Response (UPR). The validation of the microarray results by qRT PCR and Western Blot confirms that A0, but not cisplatin, activates two pathways of the UPR. In particular, IRE1 mRNA is up-regulated, resulting in the increased abundance of the spliced form of XBP1 mRNA that encodes for the active transcription factor. Moreover, the translation initiator complex subunit eIF2alpha is rapidly phosphorylated, with the consequent attenuation of protein synthesis and the concomitant preferential translation of the pro-death ER stress responsive proteins ATF4, CHOP and GADD34. In conclusion, A0 kills sensitive cancer cells through the triggering of ER stress, inducing a paraptotic process. Therefore, the copper complex may constitute a novel device to overcome apoptosis resistance.