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

Abstract

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.

Keywords: D-limonene; Drug–target–pathway network; Gene analysis; HepG2; MCF-7; Metformin.

Fig. 1

a The compound–target network of metformin and D-limonene. The blue nodes represent target genes and the pink nodes represent compounds, the edges are the interactions of targets and their related pathways. b The target–pathway network of metformin and D-limonene. The blue nodes represent target genes and the pink nodes represent pathways, the edges are the interactions of targets and their related pathways. c The Gene Ontology enrichment of therapy target genes. Y-axis represents significantly enriched biological process categories relative to target genes and the X-axis shows the counts of targets

Fig. 2

a The antitumor effect of metformin and D-limonene on HepG2 cells after incubation for 24 and 48 h. b The antitumor effect of metformin and D-limonene on MCF-7 cells after incubation for 24 and 48 h. c The cytotoxicity of IC₅₀ doses of metformin and D-limonene toward the normal cell line WI-38 after incubation for 48 h. The percentage of viable cells was measured by the MTT assay. met: metformin, lim: D-limonene

Fig. 3

1) CompuSyn-generated graphics based on numerical data given in Supplementary Table 4-A for HepG2 cells. 2) CompuSyn-generated graphics based on numerical data given in Supplementary Table 3-B for MCF-7 cells. a: dose–effect curves. b: median-effect plots. c: isobologram for combo: (Met + lim [2:1]). d: polygonogram at Fa = 0.9

Fig. 4

a DPPH free radical scavenging activity at different concentrations of metformin, D-limonene, and ascorbic acid. b GSH concentration in HepG2 and MCF-7 cells cultured in the basic medium as well as medium supplemented with metformin, D-limonene, and combination of both drugs (Met: Lim [2:1]) at IC₅₀ dose levels

Fig. 5

Dot plot flow cytometry analysis data. 1) Control untreated normal cells; 2) untreated HepG2 cells; 3) HepG2 cells treated with IC₅₀ dose of D-limonene; 4) HepG2 cells treated with IC₅₀ dose of metformin; 5) HepG2 cells treated with a combination of Met: Lim (2:1); 6) untreated MCF-7 cells; 7) MCF-7 cells treated with IC₅₀ dose of D-limonene; 8) MCF-7 cells treated with IC₅₀ dose of metformin; 9) MCF-7 cells treated with a combination of Met: Lim (2:1)

Fig. 6

Flow cytometric analysis data showing average percentages of viable, necrotic, early, and late apoptotic a HepG2 cells and b MCF-7 cells treated with metformin, D-limonene, and a combination of both Met:Lim (2:1) compared with untreated normal cells

Fig. 7

Flow cytometric analysis data showing average cell percentages in each phase of cell cycle stained with Propidium iodide for HepG2 cells a and MCF-7 cells b

Fig. 8

Flow cytometric histograms for cell cycle analysis stained with Propidium iodide showing the cell cycle phases P8 = Apoptosis; P7 = (G0/G1) the first gap phase; P6 = S phase; P5 = G2/M. show HepG2 cells treatment affected the cell cycle distribution and induced apoptosis. a Control untreated normal cells; b untreated HepG2 cells; c HepG2 cells treated with metformin; d HepG2 cells treated with D-limonene; e HepG2 cells treated with combination of Met: Lim (2:1); f untreated MCF-7 cells; g MCF-7 cells treated with metformin IC50; h MCF-7 cells treated with d-limonene IC50; i MCF-7 cells treated with combination of Met: Lim (2:1)