Photodynamic therapy mediates the ROS-dependent activation of the tumor prosurvival hypoxia-inducible factor 1

MARIA JULIA LAMBERTI; NATALIA B RUMIE VITTAR; VIVIANA A RIVAROLA

Cordoba

Congreso; 16th International Congress on Photobiology; 2014

Grupo Argentino de Fotobiología

Photodynamic therapy (PDT), a promising treatment for cancer, involves the localization of a photosensitizer (PS) in the target tissue before illumination using an appropriate wavelength. On illumination, a cascade of photochemical events results in the generation of cytotoxic reactive oxygen species (ROS), which causes tumor destruction. As PDT photochemistry consumes oxygen, it can also create rapid and severe transient tissue hypoxia [1]. Hypoxia-inducible factor 1 (HIF-1) plays an important role in the pleiotropic response observed under low oxygen pressure and drives hypoxic gene expression changes that are adaptive for cells exposed to a reduced-oxygen environment [2]. Thus, we aimed to examine whether a relationship exists between oxidative stress induced by PDT and HIF-1 protein activity in photosensitized colorrectal cancer. In order to recapitule tumor architecture of the respective original tumor, we have previously developed multicellular three-dimensional spheroids which comprise a normoxic outer shell, surrounding a hypoxic core. In this work, we studied photodynamic effects using Me-ALA as PS in SW480 human colorrectal spheroids. PDT-ROS generation was detected 30 min post treatment using the non-fluorescent probe H2DCFDA by fluorescence microscopy. We showed that ROS level was significantly higher only in lethally photosensitized spheroids. To examine the expression of HIF-1 in response to oxidative stress, we established SW480 cells transfected with a plasmid consisting of a hypoxia response element (HRE) promoter and a downstream gene encoding for green fluorescent protein (SW480-HRE). We observed that enhanced ROS generation was concomitant with an induction of HIF transcriptional activity at 3 h and 12 h after PDT, monitoring GFP expression by flow cytometry. Moreover, addition of ROS-scavengers (N-acetyl cysteine) abrogated the PDT-mediated HIF-1 upregulation. In conclusion, we have established that PDT induces significant activation of the HIF-1 pathway due to oxidative stress, thereby HIF-1 would trigger adaptive mechanisms to ensure cell survival.