Role of reactive oxygen species in calcium signalling in hypoxia-induced epithelial-mesenchymal transition [Poster]

Introduction: Hypoxia is a hallmark of the cancer microenvironment and induces epithelial-mesenchymal transition (EMT) in breast cancer cells. EMT describes the transition of breast cancer cells into a more invasive phenotype. Hypoxia has been reported to cause changes in calcium (Ca²⁺) signalling in a variety of cell types including osteosarcoma and HEK293 cells, possibly through the production of reactive oxygen species (ROS), however, this pathway has not been studied in breast cancer cells.

Aims: To investigate the role of hypoxia-mediated increases in ROS in the induction of EMT and altered Ca2+ signalling in MDA-MB-468 breast cancer cells.

Methods: MDA-MB-468 breast cancer cells were incubated for 24 h at 1% O₂ to induce hypoxia. ROS increases were assessed using a cell-permeable fluorogenic probe 2´,7´-dichlorodihydrofluorescin diacetate (DCFH-DA). Real time RT-PCR was used to assess the mRNA levels of 50 calcium channels and transporters in MDA-MB-468 cells in the presence and absence of hypoxia.

Results: Hypoxia increased levels of the EMT markers vimentin and N-cadherin, and increased intracellular ROS levels. Five Ca²⁺ permeable channels belonging to different classes were identified as altered by hypoxia in MDAMB- 468 breast cancer cells. Chelation of ROS with 10 mM N-acetylcysteine (NAC) significantly changed the expression pattern of these five selected Ca²⁺ channels as well as the EMT marker N-cadherin.

Discussion: These results suggest an important role of ROS in hypoxia-mediated EMT in breast cancer cells and indicate that such changes are associated with alterations in the expression of specific Ca²⁺ permeable ion channels.