A study of the role of apoptotic cell death and cell cycle events mediating the mechanism of action of 6-hydroxycoumarin-3-carboxylatosilver in human malignant hepatic cells

Previously our research group has studied the anti-proliferative effects of a series of hydroxylated derivatives and silver (I) complexes of coumarin-3-carboxylic acid (C-3-COOH) using two human-derived carcinoma cell lines (A-498 and Hep-G2). Results obtained suggested that both hydroxylation and complexation with silver served to significantly augment the cytotoxic properties of C-3-COOH, to yield a compound, namely 6-hydroxycoumarin-3-carboxylatosilver (6-OH-C-COO-Ag) which could act as a potent and cyto-selective agent, capable of killing cancer cells, and with limited toxicity to cells derived from normal tissue. Here we seek to expand on these findings by probing the molecular mechanism underlying this effect. Results from cytological staining clearly illustrated cellular changes consistent with the induction of apoptotic cell death and which occurred 24 h post-drug-treatment. Additionally, electrophoretic analysis of genomic DNA showed the presence of a ladder pattern, characteristic of apoptotic cell death. This result was subsequently confirmed using a selection of biochemical assays, where increased activity of pro-apoptotic caspases 3 and 9, and increased cleavage of poly(ADP-ribose)-polymerase protein (PARP) were observed. This result was further underpinned by the appearance of a sub-G₁ peak, representing hypo-diploid cells, using flow cytometric analysis. Furthermore, 6-OH-C-COO-Ag was seen to function through an alteration in the percentage of cells entering the G₀/G₁ phase of cell cycle. Consequently, 6-OH-C-COO-Ag has been shown to a more potent and selective anti-cancer agent than cisplatin, capable of altering key biochemical events leading to the execution of apoptotic cell death as early as 24 h post-treatment, suggesting that it may represent a novel therapeutic agent for the safe and effective treatment of cancer in man.