Immunoblots were stripped and reprobed with release from mitochondria, thereby initiating intrinsic apoptosis. et al., 2001; Asanuma et al., 2005; Bilancio et al., 2006; Xia et al., 2006). YM-155 is a novel survivin suppressant that is Alisol B 23-acetate currently in clinical trials (Kummar et al., 2009; Satoh et al., 2009). We recently showed that YM-155 suppressed survivin expression, with little effect on the expression levels of other IAP family members and inhibited growth and viability of certain glioma cell lines, in addition to downregulating myeloid cell leukemia sequence 1 (Mcl-1) levels (Jane et al., 2013). Recent studies showed that upregulation of survivin by gene transfer enhanced resistance to TRAIL-induced apoptosis (Kim et al., 2011; Raviv et al., 2011), whereas transfection with survivin antisense enhanced sensitivity to TRAIL-induced apoptosis (Li et al., 2005; Azuhata et al., 2006). Because Mcl-1 is also a critical mediator Rabbit Polyclonal to PITPNB of cellular resistance to various anticancer therapies, including suppression of TRAIL-induced cell death (Kobayashi et al., 2005; Ricci et al., 2007; Kim et al., 2008; Oh et al., 2012), we questioned Alisol B 23-acetate whether YM-155 could sensitize resistant glioma cells to TRAIL, either by inhibition of survivin or Mcl-1 or both. In this study, we observed YM-155 sensitized glioma cells to TRAIL by promoting signaling through both the intrinsic and extrinsic apoptotic pathways. Our results demonstrate that therapeutic agents that downregulate Mcl-1 or survivin may promote the efficacy of TRAIL in the clinical setting. Materials and Methods Cell Lines. The established malignant glioma cell lines U87, U373, LN229, A172, and T98G were obtained from the American Type Culture Collection (Manassas, VA). LN18, LNZ428, and LNZ308 were provided by Dr. Nicolas de Tribolet (Lausanne, Switzerland). Human astrocytes (HAs) and growth media were obtained from ScienCell Research Laboratories (Carlsbad, CA). Cell culture conditions of these cell lines were as previously described (Jane et al., 2011, 2013; Premkumar et al., 2012). Reagents and Antibodies. Soluble human recombinant SuperKillerTRAIL (referred to as TRAIL in this article) was purchased from Enzo Biochemicals (Enzo Life Sciences, Farmingdale, NY). YM-155 was purchased from Chemie Tek (Indianapolis, IN). Caspase inhibitors (z-VAD-fmk, z-IETD-fmk, z-DEVD-fmk, and z-LEHD-fmk) were purchased from R&D Systems (Minneapolis, MN). The following antibodies were used: Mcl-1 (#4572), Bak (3814), Bax (#2774), Bid (#2002), cytochrome (#4280), cleaved poly-ADP-ribose polymerase Alisol B 23-acetate (PARP, #9546), cleaved caspase-3 (#9664), cleaved caspase-8 (#9496), cleaved caspase-9 (#9501), and for 15 minutes, supernatants were isolated, and protein was quantified using protein assay reagent (Pierce Chemical, Rockford, IL). Equal amounts of protein were separated by Alisol B 23-acetate SDS-PAGE and electrotransferred onto a nylon membrane (Invitrogen). Nonspecific antibody binding was blocked by incubation of the membranes with 4% bovine serum albumin in Tris-buffered saline (TBS)/Tween 20 (0.1%). The membranes were incubated with primary antibody overnight at 4C, washed in TBS/Tween 20, and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody in TBS/Tween 20 at room temperature for 1 hour. Proteins were visualized by Western blot chemiluminescence reagent (Cell Signaling). Where indicated, Alisol B 23-acetate the membranes were reprobed with antibodies against test. Differences were considered significant at values 0.05. Results Differential Apoptotic Responses of Glioma Cell Lines to TRAIL. Our recent study demonstrated a wide range of TRAIL sensitivity to apoptosis induction in glioma cell lines (Jane et al., 2011). As shown in Fig. 1A, annexin V/PI flow cytometry analysis clearly demonstrated that the percentage of apoptotic cells was increased to 80% when LN18 and T98G (TRAIL-sensitive) cells were treated with TRAIL (with dramatic apoptotic responses to concentrations as low as 5 ng/ml for 24 hours), whereas U373 and LNZ308 cells were resistant to TRAIL (12% cell death at 50 ng/ml TRAIL). Western blot analyses [time-course (Fig. 1B) and dose-response (Fig. 1C) studies] clearly demonstrate activation of the caspase cascade with cleavage of the initiator caspase-8 and the main effector caspase-3 in LN18 and T98G (TRAIL-sensitive).