Efficacy of metformin and electrical pulses in breast cancer MDA-MB-231 cells

Abstract

Aim: Triple-negative breast cancer (TNBC) is a very aggressive subset of breast cancer, with limited treatment options, due to the lack of three commonly targeted receptors, which merits the need for novel treatments for TNBC. Towards this need, the use of metformin (Met), the most widely used type-2 diabetes drug worldwide, was explored as a repurposed anticancer agent. Cancer being a metabolic disease, the modulation of two crucial metabolites, glucose, and reactive oxygen species (ROS), is studied in MDA-MB-231 TNBC cells, using Met in the presence of electrical pulses (EP) to enhance the drug efficacy.

Methods: MDA-MB-231, human TNBC cells were treated with Met in the presence of EP, with various concentrations Met of 1 mmol/L, 2.5 mmol/L, 5 mmol/L, and 10 mmol/L. EP of 500 V/cm, 800 V/cm, and 1,000 V/cm (with a pulse width of 100 µs at 1 s intervals) were applied to TNBC and the impact of these two treatments was studied. Various assays, including cell viability, microscopic inspection, glucose, ROS, and wound healing assay, were performed to characterize the response of the cells to the combination treatment.

Results: Combining 1,000 V/cm with 5 mmol/L Met yielded cell viability as low as 42.6% at 24 h. The glucose level was reduced by 5.60-fold and the ROS levels were increased by 9.56-fold compared to the control, leading to apoptotic cell death.

Conclusions: The results indicate the enhanced anticancer effect of Met in the presence of electric pulses. The cell growth is inhibited by suppressing glucose levels and elevated ROS. This shows a synergistic interplay between electroporation, Met, glucose, and ROS metabolic alterations. The results show promises for combinational therapy in TNBC patients.

Keywords: MDA-MB-231 cells; Metformin; cell migration; electroporation; glucose; reactive oxygen species; triple-negative breast cancer; wound healing assay.

Figure 1

Illustration of the experimental design flow and the scope of the research work. Double-sided arrow: pulse width; ROS: reactive oxygen species

Figure 2

Characterization of MDA-MB-231 cell line. (A) Microscopic image (10×); (B) the Gaussian distribution for the cell diameter (mean: 18 µm) of the untreated MDA-MB-231 cells. Avg: average

Figure 3

Chemical composition and structure of Met hydrochloride [76]

Figure 4

Met dosage curve for MDA-MB-231 cells at 24 h. The same letters indicate ns difference between the treatments, whereas different letters indicate a significant difference (^* P < 0.05). w.r.t.: with respect to

Figure 5

Cell viability assay on MDA-MB-231 at different EP conditions (500 V/cm, 800 V/cm, 1,000 V/cm, 100 µs, 1 s interval). (A) Viability percentage for EP only and EP + 5 mmol/L Met. The same letters indicate ns difference between the treatments, whereas different letters indicate a significant difference (^*** P < 0.001); (B) the heatmap plot depicting the statistical difference in cell viability observed between EP only and EP + 5 mmol/L Met counterpart. w.r.t.: with respect to

Figure 6

Microscopic image inspection of MDA-MB-231 cells at 24 h for control, 1,000 V/cm, 5 mmol/L Met and 1,000 V/cm + 5 mmol/L Met

Figure 7

Glucose assay for various treatments on MDA-MB-231 cells after 24 h of treatment. The same letters indicate ns difference between the treatments, whereas different letters indicate a significant difference (^*** P < 0.001; ^**** P < 0.0001)

Figure 8

ROS assay for various treatments in MDA-MB-231 cells at 24 h. The same letters indicate ns difference between the treatments, whereas different letters indicate a significant difference (^**** P < 0.0001)

Figure 9

Wound healing assay in MDA-MB-231 cells at t = 0 h and t = 24 h for control, 5 mmol/L Met, 1,000 V/cm + 5mmol/L. (A) Microscopic image; (B) cell-free area (mm²) quantified using ImageJ

Figure 10

Illustration of the overview of the plausible mechanism of action for Met in the presence of electric pulses. It shows the effective entering of Met into the cell via the OCT and additional pores created by EP (at 1,000 V/cm). Met promotes the conversion of ATP to adenosine diphosphate (ADP) and AMP, thereby lowering mitochondrial energy production. Met’s impact on gluconeogenesis is highlighted by showing the inhibition of converting pyruvate to glucose. By impeding this process, Met contributes to decreased glucose levels and elevated ROS levels, creating an overall energy stress situation within the cell, ultimately leading to cell death. mGPD: mitochondrial glycerol-3-phosphate dehydrogenase; NAD⁺: depleted nicotinamide adenine dinucleotide