Combination of metformin and gallic acid induces autophagy and apoptosis in human breast cancer cells

Abstract

Background and purpose: Breast cancer is the most common type of cancer and one of the major causes of death among women. Many reports propose gallic acid as a candidate for cancer treatment due to its biological and medicinal effects as well as its antioxidant properties. This study aimed to assess the effects of metformin and gallic acid on human breast cancer (MCF-7) and normal (MCF-10) cell lines.

Experimental approach: MCF7 and MCF-10 cells were treated with various concentrations of metformin, gallic acid, and their combination. Cell proliferation, reactive oxygen species (ROS), as well as cell cycle arrest were measured. Autophagy induction was assessed using western blot analysis.

Findings/results: Metformin and gallic acid did not cause toxicity in normal cells. They had a stronger combined impact on ROS induction. Metformin and Gallic acid resulted in cell cycle arrest in the sub-G1 phase with G1 and S phase arrest, respectively. Increased levels of LC3 and Beclin-1 markers along with decreased P62 markers were observed in cancerous cells, which is consistent with the anticancer properties of metformin and gallic acid.

Conclusion and implications: The effects of metformin and gallic acid on cancerous cells indicate the positive impact of their combination in treating human breast cancer.

Keywords: Apoptosis; Autophagy; Breast cancer; Gallic acid; Metformin; ROS.

Fig. 1

Effect of metformin, gallic acid, and their combinations on the viability of MCF-7 and MCF-10 cell lines. Cell viability was measured using an MTT assay. MCF-7 cells were treated for 48 h with (A) metformin (6.25, 12.5, 25, 50, and 100 mM); (B) gallic acid (15, 30, 60, 90, and 120 μg/mL); or (C) metformin (35 mM) and gallic acid, alone, or their combination. (D) MCF-10 cells were treated for 48 h with (D) metformin (6.25, 12.5, 25, 50, and 100 mM); (E) gallic acid (15, 30, 60, 90, and 120 μg/mL); or (F) metformin (35 mM) and gallic acid, alone, or their combination. Data are shown as a percentage of three independent tests (mean ± SEM). Met, Metformin; Gal, gallic acid. ^*P < 0.05 and ^***P < 0.001 indicate significant differences compared with the control group; ^#P < 0.05 and ^###P < 0.001 versus the group treated with metformin at 35 mM (Met); and ^$$$P < 0.001 between the defined groups.

Fig. 2

The effect of different concentrations of metformin, gallic acid, and their combinations on the production of reactive oxygen species. MCF-7 cells were treated with metformin (35 mM) and gallic acid (15 and 30 μg/mL), alone or in combination, for 48 h. Data are shown as a percentage of three independent tests (mean ± SEM). Met, Metformin; Gal, gallic acid. ^***P < 0.001 indicates significant differences compared with the control group; ^###P < 0.001 versus the group treated with metformin at 35 mM (Met); and ^$$$P < 0.001 between the defined groups.

Fig. 3

The effect of metformin (35 mM) and gallic acid (15 and 30 μg/mL), alone or in combination on the cell cycle of MCF-7 cells. (A) Cell cycle analysis histograms and (B) the percentage of different cell-cycle phases. Data are shown as three independent tests (mean ± SEM). Met, Metformin; Gal, gallic acid; PI, propidium iodide.

Fig. 4

The effect of metformin (35 mM) and gallic acid (15 and 30 μg/mL), alone or in combination, on autophagy. (A) Representative images from the western blot analysis of LC3-II, Beclin-1, and P62 proteins in MCF-7 cells. GAPDH was used as a loading control. Western blotting analysis of the expression of (B) LC3-II, (C) Beclin-1, and (D) P62 proteins in treated MCF-7 cells. Protein expression has been calculated in proportion to the control group. Data are shown as a percentage of three independent tests (mean ± SEM). Met, Metformin; Gal, gallic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ^*P < 0.05 and ^***P < 0.001 indicate significant differences compared with the control group; ^#P < 0.05 and ^###P < 0.001 versus the group treated with metformin at 35 mM (Met); and ^$$$P < 0.001 between the defined groups.