An evaluation of the effects of metal complexes on cell death signalling networks in breast cancer cell lines

Abstract

Despite the successful treatment of breast cancer, the prognosis for patients with triple-negative breast cancer (TNBC) remains poor. This type of breast cancer is characterised by the absence of receptors for oestrogen, progesterone, and the human epidermal growth factor receptor-2, rendering targeted therapies ineffective and highlighting the need for alternative treatment options. Alternative metal-based complexes, including copper and gold complexes, have been investigated for their potential anticancer properties. This study explored the efficacy of a small library of phenanthroline- and theophylline-based copper complexes, an 8-aminoquinoline-naphthyl copper complex, and gold(I) ferrocenyl-substituted 1,2,3-triazol-5-ylidene complexes, against a triple- negative breast cancer cell line compared to a mammary epithelial cell line. Doxorubicin and staurosporine were used as positive controls throughout the study. MDA-MB-231 breast cancer cells and MCF-10A mammary cells were grown in culture, and the cytotoxicity of the metal complexes with and without haemoxygenas-1 inhibitor, OB24,(1-[[2-[2- (4-Bromophenyl)ethyl]-1,3-dioxolan-2-yl]methyl]-1H-imidazolehydrochloride) was evaluated using a spectrophotometric method based on the reduction of a tetrazolium salt to a formazan product by viable cells. Morphological changes were assessed with fluorescent microscopy to rule out necrosis. Mitochondrial function was determined by measuring the level of the reactive oxygen species and mitochondrial membrane potential. Apoptosis-related assays such as annexin V binding, caspase-3/7, caspase-8, and caspase-9 activity confirmed apoptotic cell death. Changes in the expression levels of apoptosis regulatory proteins were identified using an apoptotic proteome array profiler. Immunofluorescence detected the expression and localization of phosphorylated p53 serine 15 (phospho-p53(s15)), the cyclin-dependent kinase inhibitor p21, nuclear factor-kappa B-inducing kinase (NIK), and haemoxygenase-1 (HMOX-1). The Western blot assay validated HMOX-1 expression and its molecular weight. AuPPh₃(trz-Fc-Dipp), CuPhenTh₂, and Cu8AqN complexes were most potent with IC50 values below 5 μM in MDA-MB-231 cells. CuPhenTh₂ and Cu8AqN showed selectivity toward MDA- MB-231 cells compared to the MCF-10A cells, as indicated by their lower IC50 values. iv Doxorubicin was less potent in MDA-MB-231 cells, with an IC50 value of 8.75 ± 0.88 μM, and was cytotoxic to MCF-10A cells with an IC50 value of 2.99 ± 0.88 μM. The copper and gold complexes caused nuclear condensation and fragmentation, consistent with apoptosis. Excessive generation of reactive oxygen species indicated oxidative stress. Increased annexin V binding to phosphatidylserine and activation of caspase-3/7 confirmed apoptotic cell death. The activation of caspase-9 and caspase-8 indicated the involvement of both the intrinsic and extrinsic apoptosis pathways, respectively. Mitochondrial membrane depolarisation suggested mitochondrial dysfunction and supported the intrinsic pathway of apoptosis. CuPhenTh₂ and Cu8AqN, which were selective to cancer cells reduced the expression of inhibitor of apoptosis proteins like cIAP1, survivin, and XIAP. The expression level of Fas/CD95 was decreased indicative of inhibition of canonical nuclear factor kappa B (NF-κB) signalling and the increased expression of NIK suggested the activation of the non-canonical NF-κB pathway. Increased nuclear phospho-p53(s15) and p21 expression indicated oxidative stress. Highly expressed HMOX-1 provided additional evidence for oxidative stress. Varied expression of hypoxia inducing factor-1α levels suggested complex modulation of HMOX-1, and its involvement in treatment response. Western blots confirmed the high expression of full length HMOX-1 with a molecular weight of 32 kDa. Inhibition of HMOX-1 enzymatic activity using OB24 decreased the IC50 value of Cu8AqN and increased that of CuPhenTh₂, indicating that HMOX-1 plays an important role in treatment response in TNBC. Given the aggressive nature of TNBC and its resistance to standard therapies, these copper and gold complexes emerge as promising candidates with partial selectivity compared to conventional chemotherapeutic agents like doxorubicin and worthy of further preclinical evaluation. Future work assessing HMOX-1 expression by other transcriptional factors, like Nrf2, and HMOX-1 gene expression at the mRNA level would provide comprehensive evidence of HMOX-1 modulation to better understand its role in TNBC treatment and improve treatment outcomes.