Abstract 2514: Radiation-induced HIF-1α prevents apoptosis through reduction of ROS productions after irradiation in glioblastoma
Authors: Shirai Katsuyuki , Palanichamy Kamalakannan , Thirumoorthy Krishnan , Patel Disha , Gordon Nicolaus , Chakravarti Arnab 
Published: June 2012

Purpose/Objective(s): Glioblastoma is one of the most radioresistant tumors, and hypoxic regions are commonly observed. Molecular markers of hypoxia, Hypoxia inducible factor-1α (HIF-1α), a transcriptional factor, is strongly expressed in glioblastoma and associated with poor survival. Recent studies have shown that HIF-1α is induced by irradiation even if under normoxic conditions, although the mechanism still remains unclear. In this study, we firstly investigated the association radiation-induced HIF-1α with radioresistance in glioblastoma cells. Next, to investigate the function of HIF-1α, we focused Pyruvate dehydrogenase kinase (PDK1), one of the target genes of HIF-1α, which can decrease reactive oxygen species (ROS) productions in hypoxic conditions. We analyzed whether radiation-induced HIF-1α decreases ROS productions through activation of PDK1 after irradiation under normoxic conditions.

Materials/Methods: Clonogenic survival assay was performed to investigate the effect of radiation on colony-forming ability in established cell lines (U87, LN18, and LN229) and primary cell lines (VC3 and MGH8). In these cell lines, HIF-1α and PDK1 expressions were analyzed by using Western blotting after irradiation. To investigate whether HIF-1α is associated with radiation resistance, we used two HIF-1α blockade strategies: stable knockdown cell lines of HIF-1α using lentivirus-based sh-RNA and YC-1 (A.G. Scientific, Inc.), a novel HIF-1α inhibitor. These blockade treatments were evaluated by clonogenic survival assay, MTS assay (Promega), and apoptotic proteins, such as cleaved caspase-3 and cleaved PARP. To measure ROS levels, CM-H2DCFDA (Invitrogen) was used at a dose of 10 µM.

Results: Clonogenic survival assay showed that U87 and LN18 were more radioresistant than VC3, MGH8, and LN229. In this study, U87 and LN18 were defined as radioresistant cells and the others were radiosensitive cells. Under normoxic conditions radiation-induced HIF-1α was detected in radioresistant cells, but not in radiosensitive cells. Knockdown of HIF-1α decreased cell proliferation in clonogenic survival assay. YC-1 decreased the radiation-induced HIF-1α and decreased cell viability in MTS assay. These HIF-1α blockades treatments increased cleaved caspase-3 and cleaved PARP expression after irradiation. To evaluate the function of HIF-1α, PDK1 expression and ROS levels were analyzed. PDK1 was increased only in radioresistant cells that have radiation-induced HIF-1α. Knockdown of HIF-1α decreased PDK1 expressions and increased ROS levels after irradiation.

Conclusions: We demonstrated that radiation-induced HIF-1α induces PDK1 expression and decreases ROS productions, resulting in prevention of apoptosis after irradiation. Additional studies are ongoing to determine the in vivo efficacy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2514. doi:10.1158/1538-7445.AM2011-2514