The effect of copper-imidazo[1,2-a]pyridine complexes on HT-29 and DLD-1 colorectal cancer cells

Gangat, Nadia
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Colorectal cancer (CRC) is the third leading cause of cancer-related deaths. In Sub-Saharan Africa CRC is associated with late-stage diagnosis, poor prognosis and limited chemotherapeutic efficacy. Current chemotherapy for the treatment of metastatic CRC is associated with severe adverse drug reactions and therapeutic failure. This study investigates the in vitro effects of a total of 25 test compounds belonging to three different classes; nucleoside analogues, novel epidermal growth factor receptor kinase inhibitors and copper-imidazo[1,2-a]pyridines compared to positive controls camptothecin and staurosporine against two colorectal cancer cell lines. The HT-29 and DLD-1 colorectal cancer cell lines were maintained in culture and the cytotoxic potential of the compounds was determined by the MTT assay. Fluorescent stains were used to evaluate morphological changes. The potential of the active compounds to induce apoptosis was evaluated by the annexin-V binding assay, a fluorometric caspase-3/7 assay, mitochondrial membrane depolarization and a caspase-9 activity assay. In HT-29 cells, an apoptotic proteome array was used to measure expression of other apoptotic proteins. Four copper-imidazo[1,2-a]pyridines; JD46, JD47, JD49 and JD88 were found to be the most active, with IC50 values in HT-29 cells ranging between 0.8 and 8.5 µM and in DLD-1 cells between 9.0 and 12.0 µM. Morphological evaluation showed membrane blebbing and fragmented nuclei. Both cell lines showed increased binding of annexin-V to the cell surface, and an increase of caspase-3/7 activity. The active compounds caused a loss of mitochondrial membrane potential as well as an increase in caspase-9 activity. This indicates that the copper-imidazo[1,2-a]pyridines induced apoptotic cell death via the intrinsic apoptotic pathway. Compared to the DLD-1 cells, HT-29 cells displayed increased sensitivity to the copper-imidazo[1,2-a]pyridines. HT-29 was most sensitive to three of the copper-imidazo[1,2-a]pyridines; JD46, JD47 and JD88. In HT-29 cells an apoptotic proteome array shows that JD46, JD47 and JD88 decreased the presence of phosphorylated p53. The expression of the HSP32 was enhanced, while catalase was inhibited suggesting the accumulation of reactive oxygen species. Moreover, JD46, JD47 and JD88 caused a decrease in expression of several inhibitors of apoptosis proteins (IAPs) as well as the antiapoptotic proteins, Bcl-2 and Bcl-x. An increase in expression of TRAIL death receptor proteins indicates the involvement of the extrinsic apoptotic pathway. Additionally, the decrease in IAPs and tumor necrosis factor receptor expression suggested that TNFR1 induction of the anti-apoptotic NFĸB transcription factor may have been inhibited, enabling the pro-apoptotic status of HT-29 cells when treated with copper-imidazo[1,2-a]pyridines. In summary, the copper-imidazo[1,2-a]pyridines were effective inducers of apoptotic cell death through activation of the intrinsic pathway and warrants further investigation.
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements of the degree of Master of Science in Medicine, Johannesburg 2018
Gangat, Nadia (2018) The effect of Copper Imidazo [1,2 - a] pyridine complexes on HT-29 and DLD -1 colorectal cancer cells, University of the Witwatersrand, Johannesburg, <>