Medium Renewal Blocks Anti-Proliferative Effects of Metformin in Cultured MDA-MB-231 Breast Cancer Cells

Abstract

Epidemiological studies indicate that metformin, a widely used type 2 diabetes drug, might reduce breast cancer risk and mortality in patients with type 2 diabetes. Metformin might protect against breast cancer indirectly by ameliorating systemic glucose homeostasis. Alternatively, it might target breast cancer cells directly. However, experiments using MDA-MB-231 cells, a standard in vitro breast cancer model, produced inconsistent results regarding effectiveness of metformin as a direct anti-cancer agent. Metformin treatments in cultured MDA-MB-231 cells are usually performed for 48–96 hours, but protocols describing renewal of cell culture medium during these prolonged treatments are rarely reported. We determined whether medium renewal protocol might alter sensitivity of MDA-MB-231 cells treated with metformin. Using the MTS assay, BrdU incorporation and Hoechst staining we found that treatment with metformin for 48–72 hours failed to suppress viability and proliferation of MDA-MB-231 cells if low-glucose (1 g/L) medium was renewed every 24 hours. Conversely, metformin suppressed their viability and proliferation if medium was not renewed. Without renewal glucose concentration in the medium was reduced to 0.1 g/L in 72 hours, which likely explains increased sensitivity to metformin under these conditions. We also examined whether 2-deoxy-D-glucose (2-DG) reduces resistance to metformin. In the presence of 2-DG metformin reduced viability and proliferation of MDA-MB-231 cells with or without medium renewal, thus demonstrating that 2-DG reduces their resistance to metformin. In sum, we show that medium renewal blocks anti-proliferative effects of metformin during prolonged treatments in low-glucose medium. Differences in medium renewal protocols during prolonged treatments might therefore lead to apparently inconsistent results as regards effectiveness of metformin as a direct anti-cancer agent. Finally, our results indicate that co-therapy with 2-DG and metformin might provide an effective strategy to overcome metformin resistance of breast cancer cells.

Comparison of the effects of metformin on MDA-MB-231 breast cancer cells in a monolayer culture and in tumour spheroids as a function of nutrient concentrations

“…Limited availability of glucose and/or other nutrients increases the sensitivity of cancer cells to metformin [17,22,23,26,31–39,46,47]. However, excepting glucose, the role of other nutrients and their combinations on the sensitivity of MDA-MB-231 cells to metformin was not examined in detail.…”

“…Metabolic pathways of cancer cells in vitro vary also between monolayer cultures and physiologically more relevant tumour spheroids [9–12]. Nutrient availability and metabolic phenotype can modify the sensitivity of cancer cells to pharmacological compounds that target cancer cell metabolism, such as metformin [17,22,23,26,31–39,46,47]. Recently, we have shown that maintaining constant glucose concentrations by daily medium renewal blocks the effects of metformin on MDA-MB-231 cells [31].…”

“…MTS assay (Promega Corp, Fitchburg, WI, USA) was performed as described previously[31]. Briefly, upon completion of the experiment, MDA-MB-231 cells were washed with phosphate saline buffer (PBS) and placed in a serum-free RPMI-1640 medium with 4.5 g/l of glucose, 2 mM glutamine and 1 mM pyruvate.…”

“…Once activated, AMPK accelerates catabolic and inhibits anabolic processes in cancer cells, which enables survival of cancer cells in energetic crisis [29,30]. Metformin-stimulated AMPK activation in breast cancer cells is more pronounced in glucose depleted conditions [30,31], while AMPK does not mediate the effects of metformin under serine depletion [33]. The effects of depletion of other nutrients on metformin-stimulated AMPK activation are still largely unknown.…”

“…Cancer cell sensitivity to metformin in vitro is not merely an intrinsic property of cancer cells, but can be modulated by nutrient concentrations in cell culture media [17,22,23,31–36] and consequently also by medium renewal protocols [31]. Metformin suppresses proliferation of various cancer cells more effectively in medium without glucose [31,36–39] or pyruvate [22,23,33,34], while the absence of glutamine increases metformin’s effects on liver cancer cells [26]. Studies imply that the absence of several amino acids might increase the effects of metformin on cancer cells synergistically with the absence of other nutrients [22,33,35].…”