Identification of RIP1 as a critical mediator of Smac mimetic-mediated sensitization of glioblastoma cells for Drozitumab-induced apoptosis.

This study aims at evaluating the combination of the tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL)-receptor 2 (TRAIL-R2)-specific antibody Drozitumab and the Smac mimetic BV6 in preclinical glioblastoma models. To this end, the effect of BV6 and/or Drozitumab on apoptosis induction and signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures and glioblastoma stem-like cells. Here, we report that BV6 and Drozitumab synergistically induce apoptosis and reduce colony formation in several glioblastoma cell lines (combination index<0.1). Also, BV6 profoundly enhances Drozitumab-induced apoptosis in primary glioblastoma cultures and glioblastoma stem-like cells. Importantly, BV6 cooperates with Drozitumab to suppress tumor growth in two glioblastoma in vivo models including an orthotopic, intracranial mouse model, underlining the clinical relevance of these findings. Mechanistic studies reveal that BV6 and Drozitumab act in concert to trigger the formation of a cytosolic receptor-interacting protein (RIP) 1/Fas-associated via death domain (FADD)/caspase-8-containing complex and subsequent activation of caspase-8 and -3. BV6- and Drozitumab-induced apoptosis is blocked by the caspase inhibitor zVAD.fmk, pointing to caspase-dependent apoptosis. RNA interference-mediated silencing of RIP1 almost completely abolishes the BV6-conferred sensitization to Drozitumab-induced apoptosis, indicating that the synergism critically depends on RIP1 expression. In contrast, both necrostatin-1, a RIP1 kinase inhibitor, and Enbrel, a TNFα-blocking antibody, do not interfere with BV6/Drozitumab-induced apoptosis, demonstrating that apoptosis occurs independently of RIP1 kinase activity or an autocrine TNFα loop. In conclusion, the rational combination of BV6 and Drozitumab presents a promising approach to trigger apoptosis in glioblastoma, which warrants further investigation.

Smac mimetic promotes glioblastoma cancer stem-like cell differentiation by activating NF-κB.

Recently, a broader role of inhibitor of apoptosis (IAP) proteins besides their antiapoptotic functions has been described. Therefore, we investigated the effect of non-toxic concentrations of the small-molecule Smac mimetic BV6, which antagonizes IAP proteins, on differentiation of cancer stem-like cells (CSLCs) derived from primary glioblastoma (GBM) specimens. Here, we identify a novel function of BV6 in regulating differentiation of GBM CSLCs by activating NF-κB. BV6 at non-lethal doses stimulates morphological changes associated with the differentiation of GBM CSLCs. BV6 increases transcriptional activity, mRNA and protein levels of the astrocytic marker GFAP without altering expression of the neuronal marker ß-III-tubulin, indicating that BV6 induces astrocytic differentiation of GBM CSLCs. Molecular studies reveal that BV6 triggers processing of the NF-κB subunit p100 to p52, nuclear translocation of p52 and p50 and increased NF-κB DNA-binding. Intriguingly, inhibition of NF-κB by overexpression of dominant-negative IκBα super-repressor (IκBα-SR) blocks the BV6-stimulated increase in GFAP and differentiation. Interestingly, this BV6-stimulated differentiation is associated with reduced expression of stemness markers such as CD133, Nanog and Sox2 in GBM CSLCs. In contrast, BV6 does not alter cell morphology, differentiation and expression of stemness markers in non-malignant neural stem cells. Importantly, BV6 treatment reduces clonogenicity of GBM CSLCs in vitro and in vivo, suppresses their tumorigenicity in orthotopic and subcutaneous mouse models and significantly increases the survival of mice. By identifying a novel role of BV6 in promoting differentiation of GBM CSLCs, these findings provide new insights into Smac mimetic-regulated non-apoptotic functions with important implications for targeting GBM CSLCs.

RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival.

The concept of cancer stem-like cells (CSCs) has gained considerable attention in various solid tumors including glioblastoma, the most common primary brain tumor. This sub-population of tumor cells has been intensively investigated and their role in therapy resistance as well as tumor recurrence has been demonstrated. In that respect, development of therapeutic strategies that target CSCs (and possibly also the tumor bulk) appears a promising approach in patients suffering from primary brain tumors. In the present study, we utilized RNA interference (RNAi) to screen the complete human kinome and phosphatome (682 and 180 targets, respectively) in order to identify genes and pathways relevant for the survival of brain CSCs and thereby potential therapeutical targets for glioblastoma. We report of 46 putative candidates including known survival-related kinases and phosphatases. Interestingly, a number of genes identified are involved in metabolism, especially glycolysis, such as PDK1 and PKM2 and, most prominently PFKFB4. In vitro studies confirmed an essential role of PFKFB4 in the maintenance of brain CSCs. Furthermore, high PFKFB4 expression was associated with shorter survival of primary glioblastoma patients. Our findings support the importance of the glycolytic pathway in the maintenance of malignant glioma cells and brain CSCs and imply tumor metabolism as a promising therapeutic target in glioblastoma.

Ultrastructural identification of dividing cells in the adult mammalian central nervous system.

In most parts of the adult mammalian central nervous system cell division is a relatively rare event, which makes it difficult to study at the ultrastructural level. We designed a protocol for reliable ultrastructural identification of proliferating cells in a tissue volume using DNA-incorporated 5-bromo-2-deoxyuridine (BrdU) as a marker. After BrdU administration the tissue is fixed and embedded in hydrophilic resin (LR Gold) and then cut in serial 1-2 microm sections and mounted on glass slides. BrdU is detected at the light microscopic level using immunogold labeling followed by silver enhancement, according to a standard procedure. After detection of labeled nuclei the section is reembedded in resin on the same glass slide. The glass is then dissolved in hydrofluoric acid and labeled cells cut in ultrathin sections for further ultrastructural analysis. The technique was tested and refined in sections of the intestine containing numerous dividing cells and, once optimized, was then applied to identify the ultrastructure of slowly proliferating putative stem cells in the adult mouse spinal cord.

Get to know your stem cells.

Our view of the central nervous system has changed dramatically over the past few years. It is now well established that new neurons are generated continuously in adult mammals, including humans. These neurons derive from self-renewing multipotent neural stem cells. The identify of these stem cells has recently been unveiled.

Identification of a neural stem cell in the adult mammalian central nervous system.

New neurons are continuously added in specific regions of the adult mammalian central nervous system. These neurons are derived from multipotent stem cells whose identity has been enigmatic. In this work, we present evidence that ependymal cells are neural stem cells. Ependymal cells give rise to a rapidly proliferating cell type that generates neurons that migrate to the olfactory bulb. In response to spinal cord injury, ependymal cell proliferation increases dramatically to generate migratory cells that differentiate to astrocytes and participate in scar formation. These data demonstrate that ependymal cells are neural stem cells and identify a novel process in the response to central nervous system injury.

Brain uptake and anticancer activities of vincristine and vinblastine are restricted by their low cerebrovascular permeability and binding to plasma constituents in rat.

Unidirectional blood-brain barrier transfer of the lipophilic anticancer agents vincristine and vinblastine was studied in anesthetized rats, using an isolated, in situ brain perfusion technique. Drug binding to plasma constituents was also measured. Despite the high lipophilicity of these agents (the log octanol/physiological saline partition coefficient equalled 2.14 and 1.68, respectively), the cerebrovascular permeability-surface area product, PA, of vincristine in plasma was only 0.49 x 10(-4) ml s-1 g-1 for parietal cerebral cortex, whereas that of vinblastine was too low for determination. These values are similar to those of water-soluble, poorly diffusible nonelectrolytes. The PAs were significantly higher in the absence of plasma protein, being 1.24 x 10(-4) and 5.36 x 10(-4) ml s-1 g-1, respectively. Even these values, determined by brain perfusion of protein-free buffer, were lower than would be expected from the lipophilicity of the agents. The results suggest that additional factors, such as steric hindrance and molecular charge distribution, related to the chemical and geometric structure and the large size of vincristine and vinblastine (molecular weight, 825 and 814 daltons, respectively) restrict their passage across the blood-brain barrier. As a consequence of their paradoxically low permeability at the blood-brain barrier and restrictive binding to plasma and blood constituents, doses of both agents that cause significant inhibition of extracerebral Walker 256 carcinosarcoma tumor implants in rat have no effect on tumor located in the brain.

Kinetics of neutral amino acid transport across the blood-brain barrier.

Neutral amino acid (NAA) transport across the blood-brain barrier was examined in pentobarbital-anesthetized rats with an in situ brain perfusion technique. Fourteen of 16 plasma NAAs showed measurable affinity for the cerebrovascular NAA transport system. Values of the transport constants (Vmax, Km, KD) were determined for seven large NAAs from saturation studies, whereas Km values for five small NAAs were estimated from inhibition studies. These data, together with our previous work, provide a complete set of constants for prediction of NAA influx from plasma. Among the NAAs, Vmax varied at least fivefold and Km varied approximately 700 fold. The apparent affinity (1/Km) of each NAA was related linearly (r = 0.910) to the octanol/water partition coefficient, a measure of NAA side-chain hydrophobicity. Predicted influx values from transport constants and average plasma concentrations agree well with values measured using plasma perfusate. These results provide accurate new estimates of the kinetic constants that determine NAA transport across the blood-brain barrier. Furthermore, they suggest that affinity of a L-alpha-amino acid for the transport system is determined primarily by side-chain hydrophobicity.

Phenylalanine transport across the blood-brain barrier as studied with the in situ brain perfusion technique.

Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. L-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 X 10(-4) mumol/s/g for Vmax, 0.011 mumol/ml for Km, and 1.8 X 10(-4) ml/s/g for KD during perfusion with fluid that did not contain competing amino acids. D-Phenylalanine competitively inhibited L-phenylalanine transport with a Ki approximately 10-fold greater than the Km for L-phenylalanine. There were no significant regional differences in Km, KD, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. L-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by approximately 20-fold, to 0.21 mumol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.

Facilitated transport of melphalan at the rat blood-brain barrier by the large neutral amino acid carrier system.

Melphalan has been reported to be actively transported into tumor cells by two amino acid carrier systems. As amino acids are transported across cerebral capillaries by a facilitated mechanism, studies were undertaken to assess whether or not melphalan was transported similarly, and additionally to determine melphalan's plasma and brain pharmacokinetics. The brain uptake of [14C]melphalan was measured by an in situ brain perfusion technique in the anesthetized rat utilizing [14C]-melphalan. The cerebrovascular permeability-surface area product of [14C]melphalan was calculated at cold melphalan concentrations from O to 16.3 mumol/ml. The permeability-surface area product was concentration dependent and decreased from 10.8 +/- 0.6 (+/- SE) X 10(-4)S-1 at 0.02 mumol/ml melphalan to 5.4 +/- 0.3 X 10(-4)S-1 at 16.3 mumol/ml. The system became saturated at a concentration in excess of 0.1 mumol/ml. The Michaelis-Menten parameters Vmax and Km, determined by nonlinear regression analysis of the permeability-surface area product data, equaled 0.9 +/- 0.3 X 10(-4) mumol/s/g and 0.15 +/- 0.06 mumol/ml, respectively, for the saturable component of melphalan's brain uptake. The Kd of the nonsaturable component was 5.3 +/- 0.03 X 10(-4)S-1. Addition of the amino acid 1-phenylalanine to the brain perfusate inhibited the saturable component of melphalan's brain uptake. The analysis of the plasma and brain concentrations of melphalan by high-performance liquid chromatography, following i.v. melphalan administration, demonstrated that approximately 15% of the drug that was present in plasma entered the brain. These data suggest that the brain uptake of melphalan is facilitated, demonstrating concentration-dependent uptake, saturation, and inhibition, and that melphalan shares the large neutral amino acid carrier system at the blood-brain barrier.

Kinetic analysis of cerebrovascular isoleucine transport from saline and plasma.

The concentration dependence of regional isoleucine transport across the blood-brain barrier was determined in anesthetized rats with the in situ brain perfusion technique of Takasato et al. [Am. J. Physiol. 247, H484-493 (1984)]. This technique allows, for the first time, accurate measurements of cerebrovascular amino acid transport in the absence of competing amino acids using saline perfusate, and in the presence of physiological concentrations of amino acids using plasma perfusate. Cerebrovascular isoleucine transport from saline perfusate followed Michaelis-Menten saturation kinetics where Vmax = 9 - 11 X 10(-4) mumol X s-1 X g-1 and Km = 0.054-0.068 mumol X ml-1 in six brain regions. A component of nonsaturable transport was not detected in any brain region even though perfusate isoleucine concentration was increased to greater than or equal to 150 times the normal plasma concentration. Isoleucine influx during plasma perfusion was only 8% of that predicted from the saline perfusion data due to transport inhibition by competing amino acids in plasma. Competitive inhibition increased the apparent Km for isoleucine transport from plasma by greater than or equal to 24-fold to 1.5-1.7 mumol X ml-1. These data provide accurate new estimates of the kinetic constants that describe amino acid transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer-site affinity (1/Km) for isoleucine is approximately fivefold greater than previously reported with the brain uptake index technique.

Survival from acute renal failure after severe burns.

We describe a patient with 50 per cent, third degree flame burns who had a history of paint thinner inhalation for over 10 years. Moreover, chlorpromazine had been administered for the treatment of insomnia caused by chronic thinner intoxication. He developed oliguric acute renal failure soon after the burn injury, although adequate resuscitation therapy was given, and survived following frequent haemodialysis. Although survival from acute renal failure after severe burns is rare, once the diagnosis of acute renal failure has been made, haemodialysis should be instituted as early as possible. Furthermore, in a severely burnt patient with episodes of chronic and acute intoxication from organic chemicals or drugs which may have caused renal damage, acute renal failure may occur, so that careful observation is advised.