Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor–related apoptosis-inducing ligand and induces an alternative cell death pathway

Neuro-Oncology 13(11):1213 – 1224, 2011. doi:10.1093/neuonc/nor067 Advance Access publication July 13, 2011 N E U RO - O N CO LO GY Christian E. Badr, Thomas Wurdinger, Jonas Nilsson, Johanna M. Niers, Michael Whalen, Alexei Degterev, and Bakhos A. Tannous Neuroscience Center and Molecular Neurogenetics Unit, Department of Neurology (C.E.B., T.W., J.M.N., M.W., B.A.T.), Center for Molecular Imaging Research, Department of Radiology (T.W., B.A.T.), and Department of Pathology, Massachusetts General Hospital; and Program in Neuroscience, Harvard Medical School (C.E.B., T.W., J.M.N., M.W., B.A.T.), Boston, Massachusetts; Neuro-oncology Research Group, Department of Neurosurgery, VU University Medical Center, Amsterdam, Netherlands (C.E.B., T.W., J.N., J.M.N.); Department of Biochemistry, Tufts University School of Medicine, Boston, USA (A.D.) Human glioblastoma (GBM) cells are notorious for their resistance to apoptosis-inducing therapeutics. We have identified lanatoside C as a sensitizer of GBM cells to tumor necrosis factor– related apoptosis-inducing ligand (TRAIL) – induced cell death partly by upregulation of the death receptor 5. We show that lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis in a GBM xenograft model in vivo. Lanatoside C on its own serves as a therapeutic agent against GBM by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential, and early intracellular ATP depletion. In conclusion, lanatoside C sensitizes GBM cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative cell death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy to counter resistance of cancer cells to apoptosis. Keywords: cardiac glycoside, glioblastoma, lanatoside C, non-apoptotic cell death, TRAIL. Received June 15, 2010; accepted April 20, 2011. Corresponding Author: Bakhos A. Tannous, PhD, Neuroscience Center, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA, 02129 USA (). G lioblastoma (GBM) is the highest-grade (grade IV) glioma and is the number one cause of primary brain tumors.1 Because of the complexity of this disease, its high invasiveness, and the notorious resistance of GBM cells to apoptosis and conventional therapies, new treatment paradigms are highly desirable. One potent cancer-specific agent is the tumor necrosis factor– related apoptosis-inducing ligand (TRAIL), also known as APO2L or TNFSF10.2 TRAIL binds and activates the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5)3 present on cancer cells, resulting in recruitment of the adapter Fas-associated death domain (FADD) and pro-caspases 8 and 10 to their death-inducing signaling complex (DISC), subsequently resulting in the activation of the caspase-induced apoptosis pathway.4 Although TRAIL has been shown to be a promising therapeutic for various types of tumors, considerable numbers of cancer cells, including GBM cells, are resistant to TRAIL-induced apoptosis.5 – 8 Cell death, defined as the irreversible loss of plasma membrane integrity, can be classified into 3 types on the basis of morphological criteria:9 apoptosis, autophagy, and necrosis. Over the past decade, cumulative evidence suggested that necrosis could also occur in a programmed fashion, notably on activation of the death-domain receptors (DRs), while caspase activity is blocked.10,11 This additional type of cellular demise has been termed “necroptosis.”12 A clear distinction between apoptosis and necroptosis can be made because the latter induces necrotic cell # The Author(s) 2011. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: . Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor –related apoptosisinducing ligand and induces an alternative cell death pathway Badr et al.: TRAIL sensitizer and non-apoptotic cell death morphology, does not involve caspase activation, and is independent of the Bcl2 family of apoptotic regulators.12 In this study, we show that lanatoside C sensitizes glioblastoma cells to TRAIL both in cultured cells and in subcutaneous xenografts in vivo. Moreover, we show that lanatoside C on its own kills GBM cells by activating a caspase-independent cell death pathway. Activation of this alternative cell death pathway may be a useful strategy to counter apoptosis resistance in cancer cells and may be further exploited for the development of novel cancer therapeutics. Reagents The following reagents were purchased from SigmaAldrich: 3-methyladenine, bafilomycin A1, lanatoside C, and ouabain. Lanatoside C was reconstituted at 10 mg/mL (equivalent to 10 mM) in dimethyl sulfoxide (DMSO). Z-VAD-FMK, staurosporine, and rapamycin were purchased from Calbiochem. Recombinant human TRAIL (GF092) was purchased from Chemicon International. Recombinant Human TRAIL R2/TNFRSF10B Fc Chimera and Q-VD-OPh were obtained from R&D systems. Necrostatin-1 (LDN-57446) was a kind gift from Dr Greg Cuny (Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center). Cell Culture All animal studies were approved by the Massachusetts General Hospital Review Board. Initially, the maximum tolerance dose (MTD) of lanatoside C was assessed. Groups of 5 nude mice were injected with escalating doses of this drug (range, 1 – 60 mg/kg body weight) over 5 days. Control groups received the maximum amount of the vehicle DMSO. We found that the MTD of lanatoside C was 10 mg/kg body weight. One million U87-Gluc-CFP cells were implanted subcutaneously in the flanks of nude mice. One week later, mice were divided into groups of 5 mice that received intraperitoneal (i.p.) injection of either phosphate buffered saline, TRAIL (250 mg/kg body weight), lanatoside C (6 mg/kg body weight) or similar doses of both lanatoside C and TRAIL. These injections were repeated daily over 10 days. Tumor volume was monitored by both Gluc-blood assay and Gluc in vivo bioluminescence imaging, as described elsewhere.14,15 This experiment was repeated 2 times to obtain statistical significance. DNA and siRNA Transfection Cells were plated in 6-well plates (0.5 × 106), and 24 h later, transfection was performed using 2 mg of plasmid DNA and Lipofectamine 2000 (Invitrogen), in accordance with the manufacturer’s instructions. U87-Gluc-CFP cells were transfected with pBABe empty vector, pBABe-Bcl2, pBABe-Bcl-xL, or EGFP-LC316 (Addgene plasmid 11 546). To generate stable cell lines, cells were selected using hygromycin B (Invitrogen), 150 ng/mL. For siRNA experiments, U87 cells were trasfected with 100 nM of All Stars siRNA control (Qiagen) or BECN1 siRNA (Sigma) using Lipofectami (...truncated)