Wilfried Kugler

Alkylglycerol opening of the blood-brain barrier to small and large fluorescence markers in normal and C6 glioma-bearing rats and isolated rat brain capillaries.

The blood–brain barrier (BBB) represents the major impediment to successful delivery of therapeutic agents to target tissue within the central nervous system. Intracarotid alkylglycerols have been shown to increase the transfer of chemotherapeutics across the BBB. We investigated the spatial distribution of intracarotid fluorescein sodium and intravenous lissamine‐rhodamine B200 (RB 200)–albumin in the brain of normal and C6 glioma‐bearing rats after intracarotid co‐administration of 1‐O‐pentylglycerol (200 mM). To elucidate the mechanisms involved in the alkylglycerol‐mediated BBB opening, intraluminal accumulation of fluorescein isothiocyanate (FITC)–dextran 40,000 was studied in freshly isolated rat brain capillaries using confocal microscopy during incubation with different alkylglycerols. Furthermore, 1‐O‐pentylglycerol‐induced increase in delivery of methotrexate (MTX) to the brain was evaluated in nude mice. Microscopic evaluation showed a marked 1‐O‐pentylglycerol‐induced extravasation of fluorescein and RB 200–albumin in the ipsilateral normal brain. In glioma‐bearing rats, increased tissue fluorescence was found in both tumor tissue and brain surrounding tumor. Confocal microscopy revealed a time‐ and concentration‐dependent accumulation of FITC–dextran 40,000 within the lumina of isolated rat brain capillaries during incubation with 1‐O‐pentylglycerol and 2‐O‐hexyldiglycerol, indicating enhanced paracellular transfer via tight junctions. Intracarotid co‐administration of MTX and 1‐O‐pentylglycerol (200 mM) in nude mice resulted in a significant increase in MTX concentrations in the ipsilateral brain as compared to controls without 1‐O‐pentylglycerol (P<0.005). In conclusion, 1‐O‐pentylglycerol increases delivery of small and large compounds to normal brain and brain tumors and this effect is mediated at least in part by enhanced permeability of tight junctions.

Molecular basis of neurological dysfunction coupled with haemolytic anaemia in human glucose-6-phosphate isomerase (GPI) deficiency

Glucose-6-phosphate isomerase (GPI) deficiency, an autosomal recessive genetic disorder with the typical manifestation of nonspherocytic haemolytic anaemia, can be associated in some cases with neurological impairment. GPI has been found to be identical to neuroleukin (NLK), which has neurotrophic and lymphokine properties. To focus on the possible effects of GPI mutations on the central nervous system through an effect on neuroleukin activity, we analysed DNA isolated from two patients with severe GPI deficiency, one of them with additional neurological deficits, and their families. The neurologically affected patient (GPI Homburg) is compound heterozygous for a 59 A→C (H20P) and a 1016 T→C (L339P) exchange. Owing to the insertion of proline, the H20P and L339P mutations are likely to affect the folding and activity of the enzyme. In the second family studied, the two affected siblings showed no neurological symptoms. The identified mutations are 1166 A→G (H389R) and 1549 C→G (L517V), which are located at the subunit interface. We propose that mutations that lead to incorrect folding destroy both catalytic (GPI) and neurotrophic (NLK) activities, thereby leading to the observed clinical symptoms (GPI Homburg). Those alterations at the active site, however, that allow correct folding retain the neurotrophic properties of the molecule (GPI Calden).

Semaphorin-3D and semaphorin-3E inhibit the development of tumors from glioblastoma cells implanted in the cortex of the brain.

Class-3 semaphorins are secreted axon guidance factors. Some of these semaphorins have recently been characterized as suppressors of tumor progression. To determine if class-3 semaphorins can be used to inhibit the development of glioblastoma-multiforme tumors, we expressed recombinant sema-3A, 3B, 3D, 3E, 3F or 3G in U87MG glioblastoma cells. Sema3A and sema3B expressing cells contracted and changed shape persistently while cells expressing other semaphorins did not. Sema3A and sema3F differed from other semaphorins including sema3B as they also inhibited the proliferation of the cells and the formation of soft agar colonies. With the exception of sema3G and sema3B, expression of these semaphorins in U87MG cells inhibited significantly tumor development from subcutaneously implanted cells. Strong inhibition of tumor development was also observed following implantation of U87MG cells expressing each of the class-3 semaphorins in the cortex of mouse brains. Sema3D and sema3E displayed the strongest inhibitory effects and their expression in U373MG or in U87MG glioblastoma cells implanted in the brains of mice prolonged the survival of the mice by more then two folds. Furthermore, most of the mice that died prior to the end of the experiment did not develop detectable tumors and many of the mice survived to the end of the experiment. Most of the semaphorins that we have used here with the exception of sema3D were characterized previously as inhibitors of angiogenesis. Our results indicate that sema3D also functions as an inhibitor of angiogenesis and suggest that the anti-tumorigenic effects are due primarily to inhibition of tumor angiogenesis. These results indicate that class-3 semaphorins such as sema3D and sema3E could perhaps be used to treat glioblastoma patients.

Stanniocalcin 2 promotes invasion and is associated with metastatic stages in neuroblastoma

Stanniocalcin 2 (STC2) is a secreted glycoprotein of as yet unknown functions. We investigated STC2 in human neuroblastoma, the most common solid extra‐cranial tumor of infancy. In primary tumor samples, we found that expression of STC2 is associated with the metastatic Stages 4 and 4s and MYCN expression. In vitro, however, we demonstrate that cell proliferation is reduced by STC2 due to an increase in the basal apoptosis rate of the transfected cells. On the other hand, in vitro assays showed that STC2‐transfected neuroblastoma cells have an increased invasive potential and display higher activity of collagen‐degrading matrix metalloproteinase 2 (MMP2). Using experimental tumors on the chick chorioallantoic membrane (CAM), we observed that STC2 expressing cells show signs of emigration from the solid tumor and destroy blood vessels of the CAM, giving rise to massively bleeding tumors. Erosion of blood vessels was also seen when purified STC2 protein was applied on the CAM. Taken together, we demonstrate a dual role for STC2 in neuroblastoma. It reduces proliferation of tumor cells in vitro, but increases the invasive potential and induces bleeding, and thereby may facilitate early metastasis. The potential of STC2 as a surrogate marker for metastatic neuroblastoma calls for further investigation.

Intracarotid administration of short‐chain alkylglycerols for increased delivery of methotrexate to the rat brain

The intracarotid administration of alkylglycerols has been reported previously by us to be a novel strategy for increased delivery of various chemotherapeutic drugs to the normal brain and brain tumors in rats. Effectiveness and structure–activity relations of the most promising pentyl‐ and hexylglycerol derivatives have been elucidated in vivo by analyzing the transfer of methotrexate (MTX) across the blood–brain barrier (BBB) in normal rats. The effects were compared with BBB disruption using hypertonic mannitol or intracarotid infusion of bradykinin. Furthermore, toxicity of the alkylglycerols has been studied in long‐term experiments. Apart from 1‐O‐pentyldiglycerol, all alkylglycerols induced a concentration‐dependent increase in MTX delivery to the brain varying from 1.1 to more than 300‐fold compared to intra‐arterial MTX alone. Enhanced barrier permeability rapidly approached baseline values within 5 and 120 min at the latest. Chemical structure, concentration, time schedule of injections and combination of different alkylglycerols were identified as instruments suited to regulate the MTX accumulation within a wide range. Mannitol 1.4 M resulted in very high MTX levels in the brain as observed using the highest concentrations of alkylglycerols. Intracarotid infusion of bradykinin had only a minor effect on the BBB. Using 1‐O‐pentylglycerol or 2‐O‐hexyldiglycerol, both cell culture experiments and long‐term in vivo analyses including clinical, laboratory and histopathological evaluations revealed no signs of toxicity. In summary, intracarotid short‐chain alkylglycerols constitute a very effective and low toxic strategy for transient opening of the BBB to overcome the limited access of cytotoxic drugs to the brain.

Downregulation of Apaf-1 and caspase-3 by RNA interference in human glioma cells: Consequences for erucylphosphocholine-induced apoptosis

Erucylphosphocholine (ErPC) exerts strong anticancer activity in vivo and in vitroand induces apoptosis even in chemoresistant glioma cell lines. We investigated the contribution of Apaf-1 and caspase-3 to the apoptotic response to ErPC using RNA interference (RNAi) in human glioblastoma cells. We could demonstrate that human glioma cell lines are susceptible to RNAi. Apaf-1 and caspase-3 are amenable to specific small interfering RNA (siRNA)-induced degradation resulting in a reduction of protein levels to 8–33% (Apaf-1) and to 30–50% (caspase-3). Transfection of siRNA directed to Apaf-1 and caspase-3 specifically reduced caspase-3 processing induced by ErPC treatment and yielded a reduction in cells that undergo ErPC-induced apoptosis to 17–33% (Apaf-1) and to 38–50% (caspase-3). The caspase-3 siRNA experiments were corroborated in caspase-3-deficient and -reconstituted MCF-7 breast cancer cells. Survival assays and morphological observations revealed that caspase-3 reconstitution significantly sensitized MCF-7 cells to ErPC. Exploring the caspase cascade responsible for ErPC-induced apoptosis MCF-7 cells provided evidence that caspase-3 is required for the activation of caspases-2, -6 and -8 and also participates in a feedback amplification loop. Our results provide evidence that Apaf-1 and caspase-3 are major determinants of ErPC-induced apoptosis and the possible use of ErPC in a clinical setting is discussed.

Acute effects of short‐chain alkylglycerols on blood‐brain barrier properties of cultured brain endothelial cells

The blood‐brain barrier (BBB) restricts drug penetration to the brain preventing effective treatment of patients suffering from brain tumours. Intra‐arterial injection of short‐chain alkylglycerols (AGs) opens the BBB and increases delivery of molecules to rodent brain parenchyma in vivo. The mechanism underlying AG‐mediated modification of BBB permeability is still unknown. Here, we have tested the effects of AGs on barrier properties of cultured brain microvascular endothelial cells.

Erucylphosphocholine‐induced apoptosis in glioma cells: involvement of death receptor signalling and caspase activation

Erucylphosphocholine (ErPC) is a promising anti‐neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti‐cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC‐mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF‐related apoptosis‐inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFα or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant‐negative FADD construct did not abolish ErPC‐induced effects. Western blot analysis indicated that ErPC‐triggered apoptosis resulted in a time‐dependent processing of caspases‐3, ‐7, ‐8 and ‐9 into their respective active subunits. Co‐treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase‐independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.

The 18 kDa translocator protein influences angiogenesis, as well as aggressiveness, adhesion, migration, and proliferation of glioblastoma cells.

Background It is known that the mitochondrial 18 kDa translocator protein (TSPO) is present in almost all peripheral tissues and also in glial cells in the brain. TSPO levels are typically enhanced in correlation with tumorigenesis of cancer cells including glioblastoma. Relevant for angiogenesis, TSPO is also present in almost all cells of the cardiovascular system. Methods We studied the effect of TSPO knockdown by siRNA on various aspects of tumor growth of U118MG glioblastoma cells in two in-vivo models: a nude mouse model with intracerebral implants of U118MG glioblastoma cells and implantation of U118MG glioblastoma cells on the chorionallantoic membrane (CAM) of chicken embryos. In vitro, we further assayed the influence of TSPO on the invasive potential of U118MG cells. Results TSPO knockdown increased tumor growth in both in-vivo models compared with the scrambled siRNA control. Angiogenesis was also increased by TSPO knockdown as determined by a CAM assay. TSPO knockdown led to a decrease in adhesion to the proteins of the extracellular matrix, including fibronectin, collagen I, collagen IV, laminin I, and fibrinogen. TSPO knockdown also led to an enhancement in the migratory capability of U118MG cells, as determined in a modified Boyden chamber. Application of the TSPO ligand 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) at a concentration of 25 µmol/l in the in-vitro models yielded results similar to those obtained on TSPO knockdown. We found no effects of PK 11195 on TSPO protein expression. Interestingly, at low nmol/l concentrations (around 1 nmol/l), PK 11195 enhanced adhesion to collagen I, suggesting a bimodal concentration effect of PK 11195. Conclusion Intact TSPO appears to be able to counteract the invasive and angiogenic characteristics related to the aggressiveness of U118MG glioblastoma cells in vivo and in vitro.

Apoptosis Induction by Erucylphosphohomocholine via the 18 kDa Mitochondrial Translocator Protein: Implications for Cancer Treatment

Many types of cancer, for example glioblastoma, show resistance against current anti-cancer treatments. One reason is that they are not capable to effectively activate their intracellular cell death pathways. Novel treatments designed to overcome these deficiencies in cancer cells present promising concepts to eradicate chemotherapy-resistant cancer cells. One of these approaches includes the membrane seeking compound erucylphosphohomocholine (ErPC3) which is part of the latest generation of alkylphospholipid analogs developed over the last two-and-a-half decades. ErPC3 exerts potent antineoplastic effects in animal models and against established cancer cell lines including, for example, glioblastoma and different types of leukemia, while sparing their normal counterparts. Starting with a historical survey, we report here on the anticancer activity of ErPC3 and on ErPC3s established mechanisms of action. We cover the current knowledge on the induction of mitochondrial apoptosis by ErPC3, including its interaction with the 18 kDa translocator protein (TSPO). In addition we discuss other signaling pathways modulated by ErPC3. Interaction with the TSPO leads to activation of the mitochondrial apoptosis cascade. This includes cardiolipin oxidation at mitochondrial levels, collapse of the mitochondrial membrane potential, and release of cytochrome c, the initiating steps of the mitochondrial apoptosis cascade. Other pathways modulated by ErPC3 include different kinases for the PI3K/Akt/mTOR and the MAP kinase pathways. Furthermore, ErPC3s cytotoxic actions may include its effects on phosphatidylcholine synthesis to inhibit the endoplasmic reticulum enzyme CTP:phosphocholine cytidyltransferase. These basic research data hopefully will lead to effective approaches toward exploitation of ErPC3 for the treatment of cancer.