Cycling hypoxia increases U87 glioma cell radioresistance via ROS induced higher and long-term HIF-1 signal transduction activity

Abstract

Abstract. Glioblastoma multiforme (GBM) tumors are the most common type of brain tumors and resistance to radiotherapy. This study aimed to investigate the differential effect and mechanism of tumor microenvironments, cycling hypoxia and non-interrupted hypoxia, on tumor cell radiosensitivity in the human U87 glioblastoma tumor model. We exposed U87 cells and mice bearing U87 glioma to experimentally imposed cycling or non-interrupted hypoxic stress in vitro and in vivo prior to treatment with ionizing irradiation. Clonogenic survival assay and tumor growth measurements were performed to determine tumor radiosensitivity. The differential regulation of non-interrupted vs. cycling hypoxia by hypoxia-inducible factor-1 (HIF-1) and the impact of HIF-1· on hypoxia-induced radioresistance were assessed by molecular assay and RNAiknockdown technology. Our results demonstrated that cycling hypoxia induced higher and longer term HIF-1 signal transduction activity via reactive oxygen species (ROS) in U87 cells compared with non-interrupted hypoxia. Cycling hypoxiainduced HIF-1· activation reflected ROS mediated HIF-1· synthesis and stabilization, whereas non-interrupted hypoxiainduced HIF-1· activation was due to decreased HIF-1· degradation resulting from decreased prolyl hydroxylation. With regard to tumor radiosensitivity, cycling hypoxia induced more tumor cell radioresistance and a decreased response to radiotherapy in U87 cells compared with non-interrupted hypoxia. HIF-1 knockdown during in vitro and in vivo hypoxic stresses combined with radiotherapy suppressed cycling and non-interrupted hypoxia-induced radioresistance while increasing overall tumor radiosensitivity. Our results suggest that cycling hypoxia induces more radioresistance than non-interrupted hypoxia in U87 gliomas, and ROS mediated HIF-1· activation is a crucial mechanism involved in hypoxia-induced differential radioresistant in U87 gliomas.

Cycling hypoxia: A key feature of the tumor microenvironment

“…In more relevant models of cycling hypoxia (vs. one cycle of hypoxia-reoxygenation), we observed an increase in ROS production after the reoxygenation steps but not after the hypoxic periods in endothelial cells [36]. Using a similar timing of pO 2 level fluctuations, Hsieh et al (2010) also observed an increase in ROS production in U87 glioma cells [40]. Some in vivo models of cycling hypoxia have also evidenced an increase in the oxidative stress assessed by monitoring lipid peroxidation or 8-oxoguanine compared to chronic hypoxic-treated tumors or control tumors [40,56].…”

“…Several reports have shown that experimental cycling hypoxia activates HIF-1 via an increase in HIF-1 protein level despite periods of reoxygenation. This was observed in endothelial cells [36,37] as well as in cancer cells [38][39][40][41]. It is noteworthy that, in most of these reports, cells exposed to cycling hypoxia exhibited a more robust accumulation of HIF-1 than cells undergoing chronic hypoxia.…”

“…It is noteworthy that, in most of these reports, cells exposed to cycling hypoxia exhibited a more robust accumulation of HIF-1 than cells undergoing chronic hypoxia. Several mechanisms were identified including activation of protein kinase A (PKA) during the hypoxia periods [42], enhanced mitochondrial respiration as well as activation of the Akt pathway during the reoxygenation period [37] but also increased ROS production [40,41]. In addition to HIF-1, two other transcription factors have also been reported to be activated by cycling hypoxia: nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) in endothelial cells [43] and in cancer cells [41] as well as nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in A459 lung carcinoma cells [39].…”

Stabilisation and Knockdown of HIF - Two Distinct Ways Comparably Important in Radiotherapy

“…Our data derived from clonogenic survival assays of HIF-expressing hepatoma, osteosarcoma, breast adenocarcinoma and embryonal kidney cells versus their HIF-deficient counterparts are consistent with the observations of other groups on glioma, fibrosarcoma and pancreatic cancer cells [17][18][19][20]. The present results furthermore agree with the observed inverse correlation between HIF-1α expression and the prognosis for the outcome of radiotherapy [21,22].…”

Three-Dimensional In Vitro Models in Glioma Research - Focus on Spheroids

“…Features existing in these spheroids such as cell-cell contact, variation in the cell cycle distribution, diffusion effects, altered metabolism and hypoxia may influence the outcome of treatment, contributing to a better resemblance of the in vivo situation than obtained with a monolayer model (Olive & Durand, 1994;Sutherland & Durand, 1972). One feature of particular importance in these spheroids is possibly the low oxygen status being partly responsible for the increased radioresistance of the spheroid tumor cells (Blazek et al, 2007;Hsieh et al, 2010;Sutherland, 1998). Ionising radiation causes the formation of reactive oxygen species (ROS) (Brahme & Lind, 2010; and oxygen has therefore long been known to be a potent radiosensitizer (Vlashi et al, 2009).…”