ANTICANCER PROPERTY OF CHIRAL PAIRS OF TERNARY COPPER(II) COMPLEXES OF 1,10-PHENANTHROLINE AND ALANINE

Abstract

Copper complexes may serve as potential alternatives to platinum-based anticancer agents. By incorporating endogenous metal, such as copper, into metal complexes may reduce toxicity and solve other problems encountered by platinum-based antitumor drugs. A previous study had established that the chiral pairs of ternary copper(II) complex salts, [Cu(phen)(ala)(H2O)]NO3, where ala = alanine, dissociated to yield their respective [Cu(phen)(aa)(H2O)]+ and NO3- ions when dissolved in solution and these species were stable up to more than 24 hours. In this study, two pairs of optically pure chiral [Cu(phen)(ala)(H2O)]X • xH2O, where phen = 1,10-phenanthroline; X = NO3-; ala = L-alanine (L-ala) [LN] and D-alanine (D-ala) [DN]; and X = Cl-; ala = L-ala [LC] and D-ala [DC]; x = number of lattice water molecules, complex salts had been tested on MCF-7 breast cancer cells and its corresponding non-cancer cells, MCF-10A, to investigate the effect of chirality of the alanine and the change in couterion on their anticancer properties and their mechanisms of action. NCI-60 modified MTT assay and morphological study showed that the pairs of chiral ternary copper(II) compounds induced cytostatic and cytotoxic effect in a dose-dependent manner, and there was difference in cell proliferation inhibition on MCF-7 and MCF-10A cells for specific concentration range. The results also showed that chirality did affect their anti-proliferative effect on immortalised MCF-10A but not on cancer cells. However, there were insignificant differences between NO3- and Cl- pairs of the compounds towards MCF-7 and MCF-10A for 48-hour incubation. The ternary copper(II) compounds also induced dose-dependent cell population reduction towards other cancer and non-cancer cell lines, the compounds were selective towards cancer cells over normal cells. Interestingly, human breast carcinoma, MCF-7 was most responsive among the cancer cell lines tested while human hepatocellular carcinoma, HepG2, was the least sensitive towards the ternary copper(II) compounds. The results of screening of [LN] on the NCI panel of 60 human cancer cell lines did show leukaemia was the most resistant and melanoma was the most sensitive. The findings from morphological studies indicated that MCF-7 cells and MCF-10A cells treated with the ternary copper(II) compounds underwent decrease in healthy cells population and cell death by apoptosis. Apoptosis assay using Annexin V-FITC/PI double staining showed that the ternary copper(II) compounds were more selective towards MCF-7 as apoptotic cells in treated MCF-7 cells were more than those in treated immortalised breast MCF-10A cells. Cell cycle data analysis demonstrated that all the compounds suppressed MCF-7 cells growth by accumulate DNA fragments at SubG1 phase. The assay results from using 2’,7’-dicholofluorescein diacetate (DCFH-DA) showed that all the ternary copper(II) compounds induced increase in reactive oxygen species (ROS) in MCF-7 cells with increasing concentration and prolonged exposure (12 to 24 hours) compared to untreated cells. In contrast, the MCF-10A cells, treated under same conditions, showed lower overall ROS generation. The ternary copper(II) compounds induced greater mitochondrial membrane depolarisation in MCF-7 cells than in MCF-10A cells. The effect of all ternary copper(II) compounds on caspase-3/7 activity of MCF-7 cells was no significant for both 12- and 24-hour incubation but they induced distinctive but low increase of caspase-9 activity, especially at 24-hour incubation. In contrast, it was found that treating MCF-10A cells with increasing concentration of each of the ternary copper(II) compounds resulted in activation of both initiator caspase-9 and executor caspase-3/7. These results suggest that apoptosis induction in MCF-10A cells by the above compounds required activation of caspase-3/7 whereas that in MCF-7 did not, thereby implicating activation of different apoptosis pathways. Overall, these findings suggested that ternary copper(II) compounds killed the cancer cells by inducing ROS production, depolarising mitochondrial membrane, and activation of caspase-independent pathway. Therefore, the anticancer properties of the ternary copper(II) compounds involved multiple mode of actions and they also exhibited significant selectivity towards cancer cells rather than non-cancerous cells.