Bcl2L13 is a ceramide synthase inhibitor in glioblastoma.

Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13-CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use.

Spherical nucleic acid nanoparticle conjugates as an RNAi-based therapy for glioblastoma.

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many "undruggable" oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)-based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)--an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas--were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.

Glioma oncoprotein Bcl2L12 inhibits the p53 tumor suppressor.

Glioblastoma multiforme (GBM) is a lethal brain tumor characterized by intense apoptosis resistance and extensive necrosis. Bcl2L12 (for Bcl2-like 12) is a cytoplasmic and nuclear protein that is overexpressed in primary GBM and functions to inhibit post-mitochondrial apoptosis signaling. Here, we show that nuclear Bcl2L12 physically and functionally interacts with the p53 tumor suppressor, as evidenced by the capacity of Bcl2L12 to (1) enable bypass of replicative senescence without concomitant loss of p53 or p19 (Arf), (2) inhibit p53-dependent DNA damage-induced apoptosis, (3) impede the capacity of p53 to bind some of its target gene promoters, and (4) attenuate endogenous p53-directed transcriptomic changes following genotoxic stress. Correspondingly, The Cancer Genome Atlas profile and tissue protein analyses of human GBM specimens show significantly lower Bcl2L12 expression in the setting of genetic p53 pathway inactivation. Thus, Bcl2L12 is a multifunctional protein that contributes to intense therapeutic resistance of GBM through its ability to operate on two key nodes of cytoplasmic and nuclear signaling cascades.

Bipolar electrocautery: A rodent model of Sunderland third-degree nerve injury.

OBJECTIVE: To determine the Sunderland classification of a bipolar electrocautery injury. METHODS: Twenty-two rats received crush (a reproducible Sunderland second-degree injury) or bipolar electrocautery injury and were evaluated for functional, histomorphometric, and immunohistochemical recovery at 21 or 42 days. Animal experiments were performed between July 3 and December 12, 2007. Axonal regeneration and end plate reinnervation were evaluated in double transgenic cyan fluorescent protein-conjugated Thy1 and green fluorescent protein-conjugated S100 mice. RESULTS: Compared with crush injury, bipolar electrocautery injury caused greater disruption of myelin and neurofilament architecture at the injury site and decreased nerve fiber counts and percentage of neural tissue distal to the injury (P =.007). Complete functional recovery was seen after crush but not bipolar electrocautery injury. Serial live imaging demonstrated axonal regeneration at week 1 after crush and at week 3 after bipolar electrocautery injury. Qualitative assessment of motor end plate reinnervation at 42 days demonstrated complete neuromuscular end plate reinnervation in the crush group and only limited reinnervation in the bipolar electrocautery group. CONCLUSION: Bipolar electrocautery injury in a rodent model resulted in a Sunderland third-degree injury, characterized by gradual, incomplete recovery without intervention.

The short- and long-term effects of Seprafilm on peripheral nerves: a histological and functional study.

Extraneural scar reduction is an important goal in peripheral nerve microsurgery. The use of biosynthetic materials, such as Seprafilm , reduces postoperative adhesions in abdominopelvic gynecologic and orthopedic surgery. The study evaluates the safety of Seprafilm in proximity to nerve tissue in a noninjury (phase 1) and injury (phase 2) model. Phase 1 groups were: (1) sciatic nerve exposure and neurolysis (n = 15), (2) Seprafilm placement superficial to the nerve (n = 15), and (3) circumferentially wrapping Seprafilm around the nerve (n = 15). Outcome measures at 45 and 90 days included wound inspection, histomorphometry, and stereological analysis of vascularity. Phase II groups were: (1) sciatic nerve cut and repair alone (n = 15) or (2) nerve wrapped with Seprafilm (n = 15). Nerves were evaluated at 18, 32, and 42 days postoperatively, and animals underwent biweekly functional walking tracks. In phase I, no significant differences were detected between groups. In phase II, fewer perineural scar bands were seen with Seprafilm . Histomorphometric differences favoring Seprafilm at 18 days and favoring control at 42 days were noted ( P < 0.05), though no differences in functional outcomes were detected. Qualitatively less perineural scar tissue was seen when using Seprafilm . No functional or histological deleterious effects were noted from placing Seprafilm on intact nerves or cut and repaired nerves.

Processed allografts and type I collagen conduits for repair of peripheral nerve gaps.

Autografting is the gold standard in the repair of peripheral nerve injuries that are not amenable to end-to-end coaptation. However, because autografts result in donor-site defects and are a limited resource, an effective substitute would be valuable. In a rat model, we compared isografts with Integra NeuraGen (NG) nerve guides, which are a commercially available type I collagen conduit, with processed rat allografts comparable to AxoGen's Avance human decellularized allograft product. In a 14-mm sciatic nerve gap model, isograft was superior to processed allograft, which was in turn superior to NG conduit at 6 weeks postoperatively (P < 0.05 for number of myelinated fibers both at midgraft and distal to the graft). At 12 weeks, these differences were no longer apparent. In a 28-mm graft model, isografts again performed better than processed allografts at both 6 and 22 weeks; regeneration through the NG conduit was often insufficient for analysis in this long graft model. Functional tests confirmed the superiority of isografts, although processed allografts permitted successful reinnervation of distal targets not seen in the NG conduit groups. Processed allografts were inherently non-immunogenic and maintained some internal laminin structure. We conclude that, particularly in a long gap model, nerve graft alternatives fail to confer the regenerative advantages of an isograft. However, AxoGen processed allografts are superior to a currently available conduit-style nerve guide, the Integra NeuraGen. They provide an alternative for reconstruction of short nerve gaps where a conduit might otherwise be used.

The impact of motor and sensory nerve architecture on nerve regeneration.

Sensory nerve autografting is the standard of care for injuries resulting in a nerve gap. Recent work demonstrates superior regeneration with motor nerve grafts. Improved regeneration with motor grafting may be a result of the nerve's Schwann cell basal lamina tube size. Motor nerves have larger SC basal lamina tubes, which may allow more nerve fibers to cross a nerve graft repair. Architecture may partially explain the suboptimal clinical results seen with sensory nerve grafting techniques. To define the role of nerve architecture, we evaluated regeneration through acellular motor and sensory nerve grafts. Thirty-six Lewis rats underwent tibial nerve repairs with 5 mm double-cable motor or triple-cable sensory nerve isografts. Grafts were harvested and acellularized in University of Wisconsin solution. Control animals received fresh motor or sensory cable isografts. Nerves were harvested after 4 weeks and histomorphometry was performed. In 6 animals per group from the fresh motor and sensory cable graft groups, weekly walking tracks and wet muscle mass ratios were performed at 7 weeks. Histomorphometry revealed more robust nerve regeneration in both acellular and cellular motor grafts. Sensory groups showed poor regeneration with significantly decreased percent nerve, fiber count, and density (p<0.05). Walking tracks revealed a trend toward improved functional recovery in the motor group. Gastrocnemius wet muscle mass ratios show a significantly greater muscle mass recovery in the motor group (p<0.05). Nerve architecture (size of SC basal lamina tubes) plays an important role in nerve regeneration in a mixed nerve gap model.