Title
Synthesis, biological characterization and evaluation of molecular mechanisms of novel copper complexes as anticancer agents
Author
Ceyda Acilan, Zelal Adiguzel, Buse Cevatemre, Didem Karakas, Engin Ulukaya, Nádia Ribeiro, Isabel Correia, João Costa Pessoa
Year
2016
Journal
Biochimica et Biophysica Acta (BBA) - General Subjects
Abstract
To overcome the hurdles of cisplatin, majorly its toxicity and resistance, there has been extensive search for alternative anti-cancer metal-based compounds. Here, three Cu(II)-complexes, Cu(Sal-Gly)(phen), Cu(Sal-Gly)(pheamine), Cu(Sal-Gly)(phepoxy) are characterized for their interaction with DNA, cytotoxicity and mechanism of action. The binding ability of the complexes to Calf-Thymus DNA was evaluated by competition fluorescence studies with thiazole-orange, UV–Vis and circular-dichroism spectroscopic titrations. Cytotoxicity was evaluated by MTT analysis. The DNA damage was analyzed through cleavage of supercoiled DNA via agarose gel-electrophoresis, and 8-oxo-guanidine and ɣH2AX staining in cells. Apoptosis was detected via DNA condensation/fragmentation, mitochondrial membrane potential, Annexin V staining and caspase 3/7 activity. Formation of reactive oxygen species was determined by DCFDA- and GSSG/GSH-analysis. Binding constants to DNA were evaluated as 1.7 × 106 (Cu(Sal-Gly)(phen)), 2.5 × 106(Cu(Sal-Gly)(pheamine)) and 3.2 × 105 (Cu(Sal-Gly)(phepoxy)). All compounds induced DNA damage. Apoptosis was the main form of cell death. There was an increase in ROS, which is most likely responsible for the observed DNA-damage. Although the compounds were cytotoxic to all tested cancer cell lines, only Cu(Sal-Gly)(pheamine) displayed significantly lower toxicity towards non-cancer cells, its associated phenotypes differing from the other two Cu-complexes. Thus, Cu(Sal-Gly)(pheamine) was further assayed for molecular changes in response to drug treatment using a custom designed RT-qPCR array. Results showed that Harakiri was significantly upregulated. Presence of p53 was not required for apoptosis in response to Cu-complexes. These Cu-complexes, namely Cu(Sal-Gly)(pheamine), may be considered promising anticancer agents with activity in cancer cells even with deficient p53 status.
Instrument
J-720
Keywords
Circular dichroism, DNA structure, Ligand binding, Coordination chemistry, Biochemistry