Inhibitor of apoptosis (IAP) proteins are expressed at high levels in many cancers and therefore represent attractive targets for therapeutic intervention. Here, we report for the first time that the second mitochondria-derived activator of caspases (Smac) mimetic BV6 sensitizes glioblastoma cells toward Temozolomide (TMZ), the first-line chemotherapeutic agent in the treatment of glioblastoma. BV6 and TMZ synergistically reduce cell viability and trigger apoptosis in glioblastoma cells (combination index o0.4-0.8), which is accompanied by increased loss of mitochondrial-membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Analysis of the molecular mechanisms reveals that BV6 causes rapid degradation of cIAP1, leading to stabilization of NF-kB-inducing kinase and NF-kB activation. BV6-stimulated NF-kB activation is critically required for sensitization toward TMZ, as inhibition of NF-kB by overexpression of the mutant IkBa super-repressor profoundly reduces loss of mitochondrial membrane potential, cytochrome c release, caspase activation and apoptosis. Of note, BV6mediated sensitization to TMZ is not associated with increased tumor necrosis factor alpha (TNFa) production. Also, TNFa, CD95 or TRAIL-blocking antibodies or knockdown of TNFR1 have no or little effect on combination treatment-induced apoptosis. Interestingly, BV6 and TMZ cooperate to trigger the formation of a RIP1 (receptor activating protein 1)/caspase-8/FADD complex. Knockdown of RIP1 by small interfering RNA significantly reduces BV6-and TMZ-induced caspase-8 activation and apoptosis, showing that RIP1 is necessary for apoptosis induction. By demonstrating that BV6 primes glioblastoma cells for TMZ in a NF-kB-and RIP1-dependent manner, these findings build the rationale for further (pre)clinical development of Smac mimetics in combination with TMZ.

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Evasion of apoptosis contributes to radioresistance of glioblastoma, calling for novel strategies to overcome apoptosis resistance. In this study, we investigated the potential of the small molecule Smac mimetic BV6 to modulate radiosensitivity of glioblastoma cells. Here, we identify a novel proapoptotic function of NF-kB in g-irradiation-induced apoptosis of glioblastoma cells by showing, for the first time, that NF-kB is critically required for Smac mimetic-mediated radiosensitization. BV6 significantly increases g-irradiation-triggered apoptosis in several glioblastoma cell lines in a dose-and time-dependent manner. Calculation of combination index (CI) reveals that the interaction of BV6 and g-irradiation is highly synergistic (CI < 0.3). Molecular studies show that BV6 stimulates NF-kB activation, which is critical for radiosensitization, because genetic inhibition of NF-kB by overexpression of the dominant-negative superrepressor IkBa-SR significantly decreases BV6-and g-irradiation-induced apoptosis. Also, the BV6-mediated enhancement of g-irradiation-triggered caspase activation, drop of mitochondrial membrane potential, and cytochrome c release is abolished in cells overexpressing IkBa-SR. Similarly, NF-kB inhibition by ectopic expression of a kinase dead mutant of IKKb prevents the BV6-mediated sensitization for g-irradiation. The clinical relevance is underscored by experiments with primary tumor samples showing that BV6 sensitizes primary cultured glioma cells as well as glioblastoma-initiating cancer stem cells derived from surgical specimens for g-irradiation. In conclusion, we identify NF-kB as a critical mediator of Smac mimetic-conferred radiosensitization of glioblastoma cells. These results have important implications for the development of Smac mimetic-based combination protocols for radiosensitization of glioblastoma. Mol Cancer Ther; 10(10); 1867-75. Ó2011 AACR.

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The Baltic Sea is one of the world's most pollution-threatened brackish environments and limited direct consumption of fatty fish from the Baltic Sea is recommended. The use of decontaminated Baltic Sea fish raw materials as fish feed could be a strategy to recycle Baltic Sea nutrients back into food chain, while relieving pressure on aqua-feed in the growing aquaculture industry. In this study, defatted fishmeal and semi-purified fish oil from the Baltic Sea were used in fish feeds for Arctic char (Salvelinus alpinus L.). The effects of the Baltic Sea-sourced fish feeds on flesh lipid quality and fish metabolomics, compared with a standard commercial feed as a control, were determined. 1 H NMR-based metabolomics studies indicated disturbances in energy metabolism and hepatic toxicity in fish fed both crude fishmeal and crude fish oil, associated with up-regulation (IGF-I, GHR-I, PPARa, PPARb1A) and down-regulation (SREBP-1 and FAS) of hepatic genes expression. The content of n-3 long chain polyunsaturated fatty acids was not affected by the decontamination process. Thus, this short-term study demonstrates that decontaminating Baltic Sea-sourced fishmeal and fish oil reduces adverse effects in Arctic char.Practical applications: Decontaminated fish materials from the Baltic Sea were shown to be promising feed ingredients for Arctic char (Salvelinus alpinus L.) compared with untreated Baltic Sea-sourced fish feed, which induced changes in fish physiology associated with energy metabolism and hepatotoxicity. Baltic Sea-sourced fish materials containing high levels of long chain polyunsaturated fatty acids are valuable feed ingredients.