Cyclen tetrahydrochloride induces necroptosis via oxidative and nitrosative stress in mcf-7 and MDA-MB breast cancer cells respectively.

Abstract

Introduction: Breast cancer (BC) is a heterogeneous disease categorised based on the availability of specific female hormone receptors and is thus predominantly associated with female mortality and morbidity globally. The progression of BC and conventional treatments are subtype-specific, producing immunocompromising effects often linked to oxidative stress and cell death pathways, including necroptosis. A promising therapeutic synthetic compound 1,4,7,10-tetraazacyclododecane (cyclen) tetrahydrochloride is a divalent metal chelator that exhibits potential multi-target anti-cancer activities. It harbours high binding affinities toward transition-metal ions or cations, an advantageous pathway to be explored in biomedical research. Aim: This study aims to determine the anti-proliferative and antioxidant mechanisms of cyclen tetrahydrochloride in MCF-7 and MDA-MB-231 human breast cancer cells. Methods: The MTT assay assessed the cell viability of MCF-7 and MDA-MB-231 cells following exposure to cyclen tetrahydrochloride (0-1000μM) for 48 hours. Luminometric analysis of ATP and Δm ascertained mitochondrial integrity. Cells were assayed for free radical production (TBARS and NOS assays), while reactive nitrogen species (RNS) were verified by western blotting for iNOS. The antioxidant response was evaluated luminometrically (GSH) and by western blotting (Nrf2 and SOD2). In addition, signalling and cell death pathways activated by oxidative stress (caspases and externalised phosphatidylserine) were evaluated. Cell death by necroptosis was validated by qPCR analysis of RIPK1, RIPK3, MLKL, TNF-α, NF-κB, Gpx-1 and OGG1 gene expression. Results: Cell viability decreased with increasing doses of cyclen tetrahydrochloride treatments for MCF-7 (IC50 = 168.4μM, IC20 = 41.69μM) and MDA-MB-231 (IC50 = 561μM, IC20 = 302.9μM) cells. This was associated with non-significant changes in CYP34A activity and a dissipated Δm in MCF-7 cells. Although Δm was similar to the control in MDA cells, a corresponding decrease in ATP production was noted for both cell lines (p<0.05). Significant increases in MDA concentration (p<0.05) suggested lipid peroxidation associated with ROS production, particularly in MCF-7 cells. Further evidence of increased ROS was implied by increased OGG1 gene expression and decreased GSH, suggesting that oxidative stress was induced. Nitrosative stress was not evident in MCF-7 since iNOS was downregulated, and RNS were decreased. However, cyclen tetrahydrochloride upregulated iNOS to facilitate RNS production in MDA-MB-231 cells (p<0.05) and was associated with increased NF-κB gene expression (p<0.05). An inadequate antioxidant defence was demonstrated by decreased SOD2 and Nrf2 in both cell lines and Gpx-1 gene expression was upregulated in MCF-7 cells only (p<0.05). The prevailing oxidative stress did not initiate apoptosis; caspase-8 and -9 activity were decreased in both cell lines. Although caspase 3/7 was decreased for IC20 MCF-7 cells, the IC50 induced caspase 3/7 and apoptosis was executed owing to increased PS externalisation. In MDA-MB-231 cells, caspase 3/7 increased in the IC20 only, and phosphatidylserine levels were similar to the control. Interestingly, evidence of necrotic cell death in both cell lines was presented by increased DNA fluorescence and LDH leakage (p<0.05). Thus, necroptosis was investigated as an alternate mode of cell death. Significant increases in gene expression of TNF-α (p<0.05), RIPK1 (p<0.05), RIPK3 (p<0.05), and MLKL (p<0.05) demonstrated that necroptosis was increased. Conclusion: Cyclen tetrahydrochloride induced ROS-mediated necroptosis in MCF-7 cells, but ROS and RNS facilitated necroptosis in MDA-MB-231 cells.