Comparison of the effects of metformin on MDA-MB-231 breast cancer cells in a monolayer culture and in tumor spheroids as a function of nutrient concentrations
Graphical abstract
Introduction
Triple negative breast cancer, which lacks estrogen and progesterone receptors as well as human epidermal growth factor receptor 2 (HER-2), is highly aggressive form of breast cancer with limited treatment options [1]. Altered metabolic pathways are one of the hallmarks of cancer. Increased consumption of glucose under aerobic conditions, the so-called Warburg effect, was the first metabolic alteration that was described in cancer cells [2]. In addition to altered glucose metabolism, cancer cells differ from normal cells also in consumption of other nutrients, such as amino acids and lipids. Metabolic phenotype of cancer cells depends on their intrinsic characteristics and micro-environment, which is often nutrient-depleted [3]. Under in vitro conditions, metabolic phenotype differs between the standard two-dimensional (2D) and physiologically more relevant three-dimensional (3D) cell culture, which more closely resemble a tumor [4].
Metformin is the most commonly used oral drug to treat type 2 diabetes and has potential to prevent and treat breast cancer. In type 2 diabetes, metformin ameliorates glucose homeostasis and alleviates hyperinsulinemia, thus reducing the risk factors for development of insulin-sensitive cancers. Besides its systemic action, metformin targets cancer cells directly via inhibition of complex I in mitochondrial respiratory chain [5]. Metformin-mediated inhibition of mitochondrial oxidative phosphorylation reduces cellular NAD+/NADH ratio [6,7], attenuates mitochondrial anaplerotic reactions [8,9], especially aspartate biosynthesis [6,7], and induces reductive metabolism of glutamine-derived carbon in tricarboxylic acid cycle [8,10,11]. Furthermore, metformin activates AMP-activated protein kinase (AMPK), the key regulator of cellular energy homeostasis [12]. Once activated, AMPK accelerates catabolic and inhibits anabolic processes in cancer cells, which promotes survival of cancer cells in energy crisis [13]. In breast cancer cells, glucose opposes metformin-stimulated AMPK activation [14,15], while the effects of other nutrients are still largely unknown.
Nutrient concentrations [6,7,[14], [15], [16], [17], [18], [19], [20]] and medium renewal protocols [14] can modulate the sensitivity of cancer cells to metformin in vitro. Glucose and pyruvate, block the effects of metformin in various cancer cells [6,7,14,17,19,21]. Glutamine suppresses anti-cancer effects of metformin on Huh7 liver cancer cells [10] and several amino acids might reduce its effects synergistically with other nutrients [6,18,20]. Surprisingly, the effects of nutrient combinations on metformin action on MDA-MB-231 cells, the most commonly used triple negative breast cancer cell model, are poorly defined. Notably, while metformin exerts anti-cancer effects in various 3D breast cancer cell models [[22], [23], [24]], which better simulate tumor architecture than standard cell cultures, the dependence of these effects on nutrient availability remains unknown.
Here we examined the effects of nutrient availability on the sensitivity of MDA-MB-231 cells to metformin in a standard 2D monolayer culture and in 3D tumor spheroids. We show that pyruvate, glutamine and non-essential amino acids weakly oppose the effects of metformin. Furthermore, we show that glucose protects tumor spheroids against metformin-induced disintegration. Therefore, nutrient availability must be considered when we evaluate the effects of metformin on 2D and 3D cell cultures.
Section snippets
Antibodies and reagents
Metformin was from Calbiochem (Merck Millipore). All other reagents, unless otherwise specified, were from Sigma-Aldrich.
MDA-MB-231 cell culture
MDA-MB-231 cells were from ATCC (USA). MDA-MB-231 cells were grown in RPMI-1640 medium (Genaxxon bioscience, Germany) supplemented with 4.5 g/l of glucose, 2 mM L-glutamine, 1 mM pyruvate and 10% fetal bovine serum (FBS; Sigma-Aldrich). They were maintained at 37 °C in a humidified atmosphere with 5% (v/v) CO2. Experiments were performed in RPMI-1640 (Genaxxon, custom made
Nutrient availability determines the sensitivity of MDA-MB-231 cells to metformin
To assess nutrient requirements of MDA-MB-231 cells, we grew them in the complete RPMI-1640 medium or in the nutrient-deficient RPMI-1640 medium without pyruvate, glucose or glutamine for 72 h (Fig. 1A). In all our experiments complete medium contained 2 mM glutamine, 1 mM pyruvate and 4.5 g/l of glucose. The complete and nutrient-deficient media, lacking glucose, glutamine and/or pyruvate, were supplemented with 10% FBS. The depletion of glutamine and pyruvate did not affect cell
Discussion
Metabolic phenotype, which depends on nutrient availability, can modify the sensitivity of cancer cells to metabolic drug metformin [6,7,10,[14], [15], [16], [17], [18], [19], [20]]. In vitro, metabolic phenotype can vary also between 2D monolayer culture and biologically more relevant 3D tumor spheroids [4]. For that reason, we compared the effects of three major nutrients, glucose, glutamine and pyruvate, on the sensitivity of MDA-MB-231 cells to metformin in a monolayer culture and in tumor
Acknowledgements
We thank Dr. Jasna Lojk, Suzana Semič, Gregor Bizjak, Jernej Repas and Jan Gregorec for technical assistance.
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These authors contributed equally to this work.