Ternary Copper (II) Complex Induced Apoptosis and Cell Cycle Arrest in Colorectal Cancer Cells

Abstract

Background: The lack of specificity, severe side effects, and development of drug resistance have largely limited the use of platinum-based compounds in cancer treatment. Therefore, copper complexes have emerged as potential alternatives to platinum-based compounds.

Objective: Ternary copper (II) complex incorporated with 1-10-phenanthroline and L-tyrosine was investigated for its anti-cancer effects in HT-29 colorectal cancer cells.

Methods: Cytotoxic effects of ternary copper (II) complex in HT-29 cells was evaluated using MTT assay, Real-Time Cell Analysis (RTCA) and lactate dehydrogenase (LDH) assay. Cell cycle analysis was performed using flow cytometry. Apoptosis induction was studied by Annexin V-FITC/Propidium Iodide (PI) staining and mitochondrial membrane potential analysis (JC-10 staining) using flow cytometry. Intracellular Reactive Oxygen Species (ROS) were detected by DCFH-DA assay. The expression of proteins involved in the apoptotic signalling pathway (p53, caspases, and PARP-1) was evaluated by western blot analysis.

Results: Ternary copper (II) complex reduced the cell viability of HT-29 cells in a time- and dose-dependent manner, with IC50 of 2.4 ± 0.4 and 0.8 ± 0.04 μM at 24 and 48 hours, respectively. Cell cycle analysis demonstrated induction of S-phase cell cycle arrest. Morphological evaluation and Annexin V-FITC/PI flow cytometry analysis confirmed induction of apoptosis that was further supported by cleavage and activation of caspase-8, caspase-9, caspase-3, and PARP- 1. Mutant p53 was also downregulated in a dose-dependent manner. No LDH release, mitochondrial membrane potential disruption, and ROS production were observed.

Conclusion: Ternary copper (II) complex holds great potential to be developed for colorectal cancer treatment.

Keywords: Copper complex; apoptosis; caspase; cell cycle arrest; colorectal cancer; p53.