A Mechanism of Regulation of Hypoxia-Inducible Factor 1 Alpha by Nitric Oxide

Abstract

Low tissue oxygen levels, known as hypoxia, characterize many solid cancers. Both clinical and experimental studies have demonstrated that intratumoral hypoxia is associated with malignant progression defined by increased tumour growth, metastasis and resistance to therapy. A major mechanism mediating malignant adaptive responses to hypoxia involves the activity of hypoxia-inducible factor 1 (HIF-1), a transcription factor composed of HIF-1α and HIF-1β subunits; HIF-1α is highly regulated by oxygen, such that its protein levels determine HIF-1 transcriptional activity. Accumulating evidence indicates that hypoxia-induced acquisition of malignant phenotypes is, in part, due to impaired nitric oxide (NO)-mediated activation of cyclic guanosine monophosphate (cGMP) signalling and that restoration of cGMP signalling prevents such hypoxic responses. The present study aimed to determine the downstream mechanism by which the NO/cGMP signalling regulates hypoxic responses. Using DU145 prostate cancer cells, studies were conducted to assess the effect of the NO mimetic glyceryl trinitrate (GTN) and the cGMP analogue 8-Bromo-cGMP on hypoxic accumulation of HIF-1α. Results revealed that GTN, at a concentration known to primarily activate the NO/cGMP pathway, inhibited hypoxia-induced HIF-1α protein accumulation in a time-dependent manner; 8-Bromo-cGMP mimicked the effect of GTN on HIF-1α protein while levels of HIF-1α mRNA remained unaltered by exposure to either GTN or 8-Bromo-cGMP. Furthermore, treatment of cells with the calpain (Ca2+-activated proteinase) inhibitor calpastatin attenuated the effects of GTN and 8-Bromo-cGMP on HIF-1α protein accumulation. Collectively, these findings indicate a role for NO/cGMP signalling in the regulation of HIF-1α, and hence HIF-1-mediated hypoxic responses, via a mechanism that is likely dependent on calpain activity.