Molecular interactions between metformin and D-limonene inhibit proliferation and promote apoptosis in breast and liver cancer cells

Background Cancer is a fatal disease that severely affects humans. Designing new anticancer strategies and understanding the mechanism of action of anticancer agents is imperative. Hypothesis/Purpose In this study, we evaluated the utility of metformin and D-limonene, alone or in combination, as potential anticancer therapeutics using the human liver and breast cancer cell lines HepG2 and MCF-7. Study design An integrated systems pharmacology approach is presented for illustrating the molecular interactions between metformin and D-limonene. Methods We applied a systems-based analysis to introduce a drug-target-pathway network that clarifies different mechanisms of treatment. The combination treatment of metformin and D-limonene induced apoptosis in both cell lines compared with single drug treatments, as indicated by flow cytometric and gene expression analysis. Results The mRNA expression of Bax and P53 genes were significantly upregulated while Bcl-2, iNOS, and Cox-2 were significantly downregulated in all treatment groups compared with normal cells. The percentages of late apoptotic HepG2 and MCF-7 cells were higher in all treatment groups, particularly in the combination treatment group. Calculations for the combination index (CI) revealed a synergistic effect between both drugs for HepG2 cells (CI = 0.14) and MCF-7 cells (CI = 0.22). Conclusion Our data show that metformin, D-limonene, and their combinations exerted significant antitumor effects on the cancer cell lines by inducing apoptosis and modulating the expression of apoptotic genes.