BH 3 Mimetics Reactivate Autophagic Cell Death in Anoxia-Resistant Malignant Glioma Cells 1

Here, we investigated the specific roles of Bcl-2 family members in anoxia tolerance of malignant glioma. Flow cytometry analysis of cell death in 17 glioma cell lines revealed drastic differences in their sensitivity to oxygen withdrawal (<0.1% O2). Cell death correlated with mitochondrial depolarization, cytochrome C release, and translocation of green fluorescent protein (GFP)–tagged light chain 3 to autophagosomes but occurred in the absence of caspase activation or phosphatidylserine exposure. In both sensitive and tolerant glioma cell lines, anoxia caused a significant up-regulation of BH3-only genes previously implicated in mediating anoxic cell death in other cell types (BNIP3, NIX, PUMA, and Noxa). In contrast, we detected a strong correlation between anoxia resistance and high expression levels of antiapoptotic Bcl-2 family proteins Bcl-xL, Bcl-2, and Mcl-1 that function to neutralize the proapoptotic activity of BH3-only proteins. Importantly, inhibition of both Bcl-2 and Bcl-xL with the small-molecule BH3 mimetics HA14-1 and BH3I-2′ and by RNA interference reactivated anoxia-induced autophagic cell death in previously resistant glioma cells. Our data suggest that endogenous BH3-only protein induction may not be able to compensate for the high expression of antiapoptotic Bcl-2 family proteins in anoxia-resistant astrocytomas. They also support the conjecture that BH3 mimetics may represent an exciting new approach for the treatment of malignant glioma. Neoplasia (2008) 10, 873–885 Introduction Gliomas are the most common and malignant primary brain tumors in humans and are among the most hypoxic tumors known [1–3]. Uncontrolled growth, irregularities in the architecture of blood vessels, and tumor vessel occlusion through thrombosis are the main causes of tumor hypoxia [4–8]. In human glioma, the severity of tumor hypoxia is known to strongly correlate with malignancy and World Health Organization grade [1,2]. Glioblastoma multiforme, the highest-grade glioma, is characterized by large necrotic areas within the tumor tissue, and the appearance of necrosis correlates with enhanced therapy resistance, increased invasiveness, and a worse prognosis for the patients [1,2,6]. Because hypoxia provides a selection pressure for cells more tolerant to low oxygen concentrations, adaptive mechanisms to escape hypoxia-induced cell death likely play a pivotal role in gliomagenesis and tumor resistance. Activation of the transcription factor hypoxia-inducible factor is known to play a central role in the transcriptional stress response to hypoxia [8,9]. This stress response enables cells to adapt their energy metabolism, to activate survival signaling pathways, to enhance vascular blood flow, and to increase oxygen transport [8]. However, prolonged, stringent hypoxia (anoxia) leads to mitochondrial dysfunction and Abbreviations: PI, propidium iodide; LC3, light chain 3; MOMP, mitochondrial outer membrane permeabilization; TMRM, tetramethyl rhodamine methyl ester; STS, staurosporine Address all correspondence to: Donat Kögel, PhD, Experimental Neurosurgery, Johann Wolfgang Goethe University Clinics, Theodor-Stern-Kai 7, Neuroscience Center, D-60590 Frankfurt am Main, Germany. E-mail: koegel@em.uni-frankfurt.de This study was supported by grants from the Wilhelm Sander Stiftung (grant 2005.067.1) to D.K. and from the Deutsche Forschungsgemeinschaft (PR 338/9-3 and 9/4) to J.H.M.P. and D.K. These authors contributed equally to this work. Received 14 December 2007; Revised 21 May 2008; Accepted 21 May 2008 Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00 DOI 10.1593/neo.07842 www.neoplasia.com Volume 10 Number 8 August 2008 pp. 873–885 873