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

Abstract

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.

Keywords

Breast cancer / Diclofenac sodium / Piperine / D-limonene / Reactive oxygen species / Cell cycle arrest / Apoptosis

Acknowledgments

The authors acknowledge Vellore Institute of Technology management for providing the necessary facilities to carry out this work.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Funding

The authors received no extramural funding for the study.

Authors’ contributions

SS contributed to the conception and design, acquisition of data, drafting of the article, and final approval of the manuscript. GKM contributed to the analysis of data, critical revision of the manuscript content, and final approval of the manuscript.

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