Combinatorial effect of diclofenac with piperine and D-limonene on inducing apoptosis and cell cycle arrest of breast cancer cells

Objective: To investigate the potential synergistic activity of diclofenac with piperine and D-limonene in inducing apoptosis and cell cycle arrest in breast cancer MCF-7 cells. Methods: Molecular docking study was conducted to evaluate the binding affinity of diclofenac with piperine and D-limonene against p53, Bax, and Bcl-2. The MTT assay was used to determine IC50, and the Chou-Talay method was used to determine the synergistic concentration of the combination treatment of diclofenac plus piperine and diclofenac plus D-limonene. Apoptosis detection, cell cycle arrest, reactive oxygen species production, and mitochondrial membrane potential were also investigated. Results: Diclofenac, piperine, and D-limonene showed potent binding affinity for p53, Bax, and Bcl-2. Diclofenac plus piperine and diclofenac plus D-limonene enhanced the formation of reactive oxygen species, which also had an effect on the mitochondrial membrane's integrity and caused DNA fragmentation. Diclofenac plus piperine and diclofenac plus D-limonene arrested the cells in the sub-G0 phase while drastically lowering the percentage of cells in the G2/M phase. Furthermore, the elevated apoptosis in the combined therapy was confirmed by annexin V/propidium iodide staining. Conclusions: The combined therapy prominently enhanced the anti-proliferative and apoptotic effects on MCF-7 cells compared with treatment with diclofenac, piperine, and D-limonene alone.