Hydrogen Peroxide Generation as an Underlying Response to High Extracellular Inorganic Phosphate (Pi) in Breast Cancer Cells

INTRODUCTION AND OBJECTIVES: According to the growth rate hypothesis (GRH), tumour cells have high inorganic phosphate (Pi) demands due to accelerated proliferation. Compared to healthy individuals, cancer patients present with a nearly 2.5-fold higher Pi serum concentration. In this work, we show that an increasing concentration of Pi had the opposite effect on Pi-transporters only in MDA-MB-231 when compared to other breast cell lines: MCF-7 or MCF10-A (non-tumoural breast cell line). MATERIAL AND METHODS: Breast cell lines maintained in IMDM or DMEN-F12 medium are exposed to 1 mM Pi, as stated by the manufacturer. Different concentrations of NaH2PO4 were added to the regular medium 1 h or 24 h before the assays. MCF-10A, MCF-7 and MDA-MB-231 cells (5 × 104 cells per well) were incubated at 37 °C, 5% CO2 atmosphere, for 1 h in a reaction mixture (0.5 mL) containing 116 mM NaCl or choline chloride, 0.1 mM KH2PO4 or 1 mM KH2PO4 (depending on the type of Pi transporter), 5.4 mM KCl, 5.5 mM glucose, 50 mM HEPES (pH 7.2), 0.8 mM MgCl2 and 2.5 μCi/nmol 32Pi. RESULTS AND CONCLUSION: Here, we show for the first time that high extracellular Pi concentration mediates ROS production in TNBC (MDA-MB-231). After a short-time exposure (1 h), Pi hyperpolarizes the mitochondrial membrane, increases mitochondrial ROS generation, impairs oxygen (O2) consumption and increases PKC activity. However, after 24 h Pi-exposure, the source of H2O2 seems to shift from mitochondria to an NADPH oxidase enzyme (NOX), through activation of PKC by H2O2. Exogenous-added H2O2 modulated Pi-transporters the same way as extracellular high Pi, which could be reversed by the addition of the antioxidant N-acetylcysteine (NAC). NAC was also able to abolish Pi-induced Epithelial-mesenchymal transition (EMT), migration and adhesion of MDA-MB-231. We believe that Pi transporters support part of the energy required for the metastatic processes stimulated by Pi and trigger Pi-induced H2O2 production as a signalling response to promote cell migration and adhesion.