The apoptotic effect of copper-imidazo[1,2-a]pyridine complexes on leukaemic cells

Abstract

From the total numbers of leukaemia cases globally, 78% occur in developing countries. In South Africa, haematological malignancies account for 6% of new cancer cases and 8% of cancer deaths yearly. Despite the availability of targeted therapy, current treatments for acute promyelocytic leukaemia and chronic myelocytic leukaemia are still met with challenges. Therapeutic failure due to primary and acquired drug resistance as well as severe adverse effects warrants the search for new anti-leukaemic agents. Small molecules like imidazo[1,2- a]pyridines are considered to be biologically active scaffolds. These molecules complexed to metals such as copper and platinum are being actively explored for their anti-cancer potential. A series of imidazo[1,2-a]pyridine derivatives were synthesized and complexed to either copper or zinc to form metal complexes. These imidazo[1,2-a]pyridine derivatives were evaluated for their ability to inhibit leukaemic cell growth and induce apoptosis using two leukaemc cell lines; HL-60 and K562. This part of the study identified imidazo[1,2- a]pyridines complexed with copper as having superior anti-cancer activity. Three copper imidazo[1,2-a]pyridines; JD88, JD47R and JD49R were most active against HL-60 and K562 cells at low micromolar concentrations ranging between 1.0 and 6.0 µM. Morphological evaluation indicated that cell death was caused by apoptosis. The ability of the active compounds to cause apoptosis was confirmed by the Annexin-V binding assay and the fluorimetric caspase-3/7 assay. Following this, the ability of the compounds to activate the intrinsic apoptotic pathway was investigated and confirmed with the following tests: mitochondrial membrane depolarisation, caspase-8 activity and caspase-9 activity assays. Changes to the mitochondrial membrane potential were determined with the JC-1 cationic dye. An increase in Annexin-V binding, caspase-3/7, caspase-9 activity and a loss of mitochondrial membrane potential by the copper complexes indicated activation of the intrinsic apoptotic pathway In order to better understand the cell death pathways by which these compounds cause cell death, apoptotic regulatory proteins and the apoptotic transcriptome were investigated in HL 60 and K562 leukaemic cell lines. A Proteome Profiler™ Human Apoptosis Array measured the protein expression levels of 35 apoptotic-regulatory proteins. Proteomic analysis showed an increase in cleaved caspase-3 protein, confirming apoptosis. In both cell lines, the expression of pro-apoptotic proteins Bax and Smac/DIABLO were increased. The expression of inhibitor of apoptosis proteins, XIAP, cIAP-1 and survivin, were decreased, thereby reducing the apoptotic threshold. A real-time quantitative polymerase chain reaction (RT-qPCR) array was used to quantify the mRNA levels of apoptotic regulators in the presence of the copper complexes. A decrease in Akt gene expression and nuclear factor-kappa B (NF-κB) suggested the inhibition of NF κB activity. The mRNA transcript levels of non-canonical NF-κB regulated target genes were decreased, suggesting an inhibitory effect on NF-κB inducing kinase (NIK), preventing non-canonical NF-κB activation. Furthemore, expression of various key components of tumour necrosis factor signalling like TNF-α, TRADD, TNF-R1 TNF-R2, TRAF2, TRAF3 and RIPK was suppressed. The copper complexes caused intrinsic apoptosis in both leukaemic cell lines and decreased the expression levels of key cell pro-survival proteins while increasing pro-apoptotic protein expression. This appears to be mediated by inhibiting the Akt kinase activity and the transcriptional activity of the NF-κB pathway, which may be central to the apoptotic effect of the copper complexes. These copper complexes serve as valuable tools to better understand the intricacy of the death signalling pathways in leukaemic cells. The data presented here supports the further pre-clinical evaluation of the copper-imidazo[1,2-a]pyridines. This should include evaluation of efficacy in animal models of leukaemia, acute and chronic toxicity studies and the determination of pharmacokinetic properties