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.

Alzheimer’s disease (AD) is an aging-associated neurodegenerative disorder which produces a progressive and drastic decline in memory and learning. Currently, there are no effective treatment for AD. The major pathological hallmarks of AD are intracellular neurofibrillary tangles (NFTs) formation and amyloid-β (Aβ) plaques deposition in the brain. However, Aβ-dedicated therapies were not effective at treating AD as the efficacy of Aβ therapies observed in animal models is not reflected in human clinical trials. Therefore, therapies targeting Tau have gained tremendous attention as the potential treatments for AD. Previous studies had shown that antibody against phospho-Tau serine 396/404 could prevent Tau aggregation and clearing Tau oligomers and insoluble aggregates. In this study, anti-phospho-Tau serine 396/404 single chain variable fragment (scFv) antibody was generated by using Ph.D-12 phage display library. In this process, four rounds of biopanning and amplifications were carried out. Furthermore, phage ELISA binding assay was conducted to determine binding affinity of phage in each round towards the epitope. The results indicate the phage in fourth round are highly specific towards the epitope. After fourth round of biopanning and amplification, three scFv antibodies which appeared as peptide sequence motif, namely FPLNSEENPFEL, FPLNSEENPLEL and FPLNSEENAFEL were identified. These scFv antibodies were later tested on immortalised hepatocyte, CHANG, to investigate their toxicity towards normal cells via MTT cell viability assay. The results were indicating scFvs were not toxic towards immortalised hepatocytes. The scFvs antibodies also treated on AD cell model for their efficacy in eliminating hyperphosphorylated Tau. The AD cell model (T-SH-SY5Y) was produced by transfecting neuroblastoma cells, SH-SY5Y, with 2N4R Tau-441 plasmid. A protein phosphatase inhibitor, okadaic acid (OA), was added to T-SH-SY5Y cells to induce hyperphosphorylation of Tau protein. Based on MTT cell viability results, the cell viabilities had increased after treated with 10 μM scFvs antibodies for 48 hours compared to those treated with 20 nM OA. Moreover, the pSer396 and PHF-1 expression level was also decreased after treated T-SH-SY5Y cells with 10 μM scFv antibodies. The results indicated the cytotoxic effect of hyperphosphorylated Tau was reduced or eradicated by treating the T-SH-SY5Y cells with scFv antibodies. Molecular mechanism of scFv antibodies on elimination of hyperphosphorylated Tau was investigated in this study. Autophagy is a major self-degenerative process to maintain cellular homeostasis and function. Phosphatidylinositol 3-phosphate kinase (PI3K)/AKT/mammalian target of rapamycin (m-TOR) signalling pathway is the primary pathway that regulates autophagy when cells are under certain conditions such as starvation and oxidative stress. There is vast evidence have related PI3K/AKT/m-TOR signalling pathway to AD especially Tau phosphorylation. Overall, the protein expression of PI3K, AKT and m-TOR were decreased in 10 μM scFv antibodies treated T-SH-SY5Y cells. Numerous research had claimed that downregulation for these three proteins could induced autophagy in cells. A regulator at upstream of PI3K/AKT/m-TOR signalling pathway, PTEN, was also study by using western blot analysis. The results shown that PTEN protein expression was increased scFv antibodies treated T-SH-SY5Y cells. Upregulation of PTEN protein was known to inhibit the proteins (PI3K, AKT and m-TOR) involved in downstream of the signalling pathway which ultimately induced autophagy. Hence, the scFv antibodies able to eradicate Tau hyperphosphorylation via PI3K/AKT/m-TOR signalling pathway by inducing autophagy in AD cell model. In this study, possibilities of hyperphosphorylated Tau elimination via endoplasmic reticulum (ER) stress signalling pathway in T-SH-SY5Y cells also investigated. Several studies had reported ER-stress and Tau hyperphosphorylation could be induced by each other which led to promote AD-like neurodegeneration. Protein expression level of selected proteins (IRE-1-α, Calnexin, PERK, Atg12, Beclin-1 and eIF-2α) which involved in ER-stress pathway were studies by using western blot analysis. Based on western blot analysis results, majority of the protein expression level of selected protein was decreased by treating T-SH-SY5Y cells with scFv antibodies. However, the expression level of selected protein was increased in T-SH-SY5Y treated with scFvs and its mixture for 3 hours. The results indicated the ER-stress pathway was activated in early events of scFv treatment which led to autophagy in AD cell model. In conclusion, three scFv antibodies with high affinity towards phospho-Tau serine 396/404 epitope was identified. All of them did not induced cytotoxicity on immortalised hepatocytes and able to reduce cytotoxicity of hyperphosphorylated Tau protein in AD cell model. The eradication of Tau hyperphosphorylation by these scFv antibodies can be achieved via inhibition of PI3K/AKT/m-TOR and ER-stress signalling pathway.