Vol.:(0123456789) 1 3 Apoptosis https://doi.org/10.1007/s10495-019-01547-7 Copper‑imidazo[1,2‑a]pyridines induce intrinsic apoptosis and modulate the expression of mutated p53, haem‑oxygenase‑1 and apoptotic inhibitory proteins in HT‑29 colorectal cancer cells Leonie Harmse 1  · Nadia Gangat 1  · Carla Martins‑Furness 1  · Jean Dam 2  · Charles B. de Koning 2 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Metastatic colorectal cancer responds poorly to treatment and is a leading cause of cancer related deaths. Worldwide, chemo- therapy of metastatic colorectal cancer remains plagued by poor efcacy, development of resistance and serious adverse efects. Copper-imidazo[1,2-a]pyridines were previously shown by our group to be selectively active against several cancer cell lines, with three complexes, JD46(27), JD47(29), and JD88(21), showing IC 50 values between 0.8 and 1.8 μM against HT-29 cells. Here, we report that treatment with the copper complexes resulted in fragmented nuclei suggestive of apoptotic cell death, which was confrmed by increased annexin V binding and caspase-3/7 activity. The copper complexes caused a loss of mitochondrial membrane potential and increased caspase-9 activity. The absence of caspase-8 activity indicated acti- vation of the intrinsic pathway. Proteomic analysis revealed that copper-imidazo[1,2-a]pyridines decreased the expression of phosphorylated forms of p53 [phospho-p53(S15), phospho-p53(S46) and phospho-p53(S392)]. The expression of inhibitor of apoptosis proteins, XIAP, cIAP1, livin, and the antiapoptotic proteins, Bcl-2 and Bcl-x, was decreased. HO/HMOX/HSP32, expression was notably increased, which suggested the accumulation of reactive oxygen species. Increased expression of TRAIL-R2/DR5 death receptor indicated the possible dual activation of both the extrinsic and intrinsic apoptotic pathways; however, caspase-8 activation could not be demonstrated. In conclusion, the copper-imidazo[1,2-a]pyridines were efective inducers of apoptotic cell death at low micromolar concentrations and changed the expression levels of proteins important for cell survival and cell death. These copper complexes may be useful tools to better understand the complexity of signal- ling networks in cancer cell death in response to cell stress. Keywords Colorectal cancer · Copper-imidazo[1,2-a]pyridines · Intrinsic apoptosis · p53 · Haem oxygenase-1/heat shock protein 32 · Inhibitor of apoptosis proteins Introduction Whilst colorectal cancer (CRC) diagnosed at an early stage has a good prognosis, with 5 year survival rates above 90%; metastatic CRC has a poor prognosis with a 5 year survival rate below 10% [1, 2]. Current treatment regimens of meta- static CRC include 5-fuorouracil, irinotecan, oxaliplatin and leucovorin. The concurrent administration of monoclonal antibodies like bevacizumab to VEGF and cetuximab to EGFR, increases overall survival to 30 months, [2] but at a prohibitive cost. Moreover, EGFR inhibitors are only efec- tive in patients harbouring wild type KRAS [3]. The role of copper in cancer has received considerable attention in the past decade, as a protagonist as well as an antagonist and component of small molecule drug candi- dates. [4–7]. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10495-019-01547-7) contains supplementary material, which is available to authorized users. * Leonie Harmse Leonie.Harmse@wits.ac.za 1 Division of Pharmacology, Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa 2 Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, PO Wits, Johannesburg 2050, South Africa