Induction of plasminogen activator inhibitor type-1 (PAI-1) by hypoxia and irradiation in human head and neck carcinoma cell lines

Abstract

Abstract Background Squamous cell carcinoma of the head and neck (SCCHN) often contain highly radioresistant hypoxic regions, nonetheless, radiotherapy is a common treatment modality for these tumours. Reoxygenation during fractionated radiotherapy is desired to render these hypoxic tumour regions more radiosensitive. Hypoxia additionally leads to up-regulation of PAI-1, a protein involved in tumour progression and an established prognostic marker for poor outcome. However, the impact of reoxygenation and radiation on PAI-1 levels is not yet clear. Therefore, we investigated the kinetics of PAI-1 expression and secretion after hypoxia and reoxygenation, and determined the influence of ionizing radiation on PAI-1 levels in the two human SCCHN cell lines, BHY and FaDu. Methods HIF-1α immunoblot was used to visualize the degree of hypoxia in the two cell lines. Cellular PAI-1 expression was investigated by immunofluorescence microscopy. ELISA was used to quantify relative changes in PAI-1 expression (cell lysates) and secretion (cell culture supernatants) in response to various lengths (2 – 4 h) of hypoxic exposure (2), reoxygenation (24 h, 20 % O2), and radiation (0, 2, 5 and 10 Gy). Results HIF-1α expression was induced between 2 and 24 h of hypoxic exposure. Intracellular PAI-1 expression was significantly increased in BHY and FaDu cells as early as 4 h after hypoxic exposure. A significant induction in secreted PAI-1 was seen after 12 to 24 h (BHY) and 8 to 24 h (FaDu) hypoxia, as compared to the normoxic control. A 24 h reoxygenation period caused significantly less PAI-1 secretion than a 24 h hypoxia period in FaDu cells. Irradiation led to an up-regulation of PAI-1 expression and secretion in both, BHY and FaDu cells. Conclusion Our data suggest that both, short-term (~4 – 8 h) and long-term (~20 – 24 h) hypoxic exposure could increase PAI-1 levels in SCCHN in vivo. Importantly, radiation itself could lead to PAI-1 up-regulation in head and neck tumours, whereas reoxygenation of hypoxic tumour cells during fractionated radiotherapy could counteract the increased PAI-1 levels.