Sabine Spiegl-Kreinecker

Structure-activity relationship analysis of novel derivatives of narciclasine (an Amaryllidaceae isocarbostyril derivative) as potential anticancer agents.

Narciclasine (1) is a plant growth regulator that has been previously demonstrated to be proapoptotic to cancer cells at high concentrations (> or = 1 microM). Data generated in the present study show that narciclasine displays potent antitumor effects in apoptosis-resistant as well as in apoptosis-sensitive cancer cells by impairing the organization of the actin cytoskeleton in cancer cells at concentrations that are not cytotoxic (IC(50) values of 30-90 nM). The current study further revealed that any chemical modification to the narciclasine backbone generally led to compounds of variable stability, weaker activity, or even the complete loss of antiproliferative effects in vitro. However, one hemisynthetic derivative of narciclasine, compound 7k, demonstrated by both the intravenous and oral routes higher in vivo antitumor activity in human orthotopic glioma models in mice when compared to narciclasine at nontoxic doses. Narciclasine and compound 7k may therefore be of potential use to combat brain tumors.

Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes

The poor prognosis of glioma patients is partly based on the minor success obtained from chemotherapeutic treatments. Resistance mechanisms at the tumor cell level may be, in addition to the blood–brain barrier, involved in the intrinsic chemo-insensitivity of brain tumors. We investigated the expression of the drug-transporter proteins P-glycoprotein (P-gp) and multidrug-resistance protein 1 (MRP1) in cell lines (N=24) and primary cell cultures (N=36) from neuroectodermal tumors, as well as in brain tumor extracts (N=18) and normal human astrocytes (N=1). We found that a considerable expression of P-gp was relatively rare in glioma cells, in contrast to MRP1, which was constitutively overexpressed in cells derived from astrocytomas as well as glioblastomas. Also, normal astrocytes cultured in vitro expressed high amounts of MRP1 but no detectable P-gp. Meningioma cells frequently co-expressed P-gp and MRP1, while, most of the neuroblastoma cell lines express higher P-gp but lower MRP1 levels as compared to the other tumor types. Both, a drug-exporting and a chemoprotective function of P-gp as well as MRP1 could be demonstrated in selected tumor cells by a significant upregulation of cellular 3H-daunomycin accumulation and daunomycin cytotoxicity via administration of transporter antagonists. Summing up, our data suggest that P-gp contributes to cellular resistance merely in a small subgroup of gliomas, but frequently in neuroblastomas and meningiomas. In contrast, MRP1 is demonstrated to play a constitutive role in the intrinsic chemoresistance of gliomas and their normal cell counterpart.

Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity

Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found that OP-A induced marked changes in the dynamic organization of the F-actin cytoskeleton, and inhibited the proliferation and migration of GBM cells, likely by inhibiting big conductance Ca2+-activated K+ channel (BKCa) channel activity. Moreover, our results indicated that OP-A induced paraptosis-like cell death in GBM cells, which correlated with the vacuolization, possibly brought about by the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In addition, the OP-A-induced cell death did not involve the activation of caspases. We also showed that the expression of BKCa channels colocalized with these two organelles (mitochondria and ER) was affected in this programmed cell death pathway. Thus, this study reveals a novel mechanism of action associated with the anticancer effects of OP-A, which involves the induction of paraptosis through the disruption of internal potassium ion homeostasis. Our findings offer a promising therapeutic strategy to overcome the intrinsic resistance of GBM cells to proapoptotic stimuli.

O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients.

O(6)-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (chi(2) test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76-17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45-2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.

Overexpression of the human major vault protein in astrocytic brain tumor cells

Evidence has shown that the major human vault protein (MVP), which is identical to lung resistance‐related protein (LRP), may be causally involved in a special type of multidrug resistance (MDR). The purpose of this study was to investigate the expression and cellular localization of MVP in cells derived from brain tumors and other tumors of neuroectodermal origin. Using both established cell lines (n = 22) and primary explants (n = 30), we show that a distinct overexpression of the MVP gene at the mRNA (RT‐PCR) and protein (Western blot) levels is a characteristic feature of cells derived from astrocytic brain tumors. Primary cultures obtained from meningioma specimens also expressed high MVP levels, in contrast to neuroblastoma and medulloblastoma cells, which rarely contained detectable amounts of MVP. Normal human astrocytes cultured in vitro expressed MVP, although at low amounts compared with most malignant cell types. Basal MVP expression correlated with resistance against diverse antineoplastic drugs including anthracyclins, cisplatin and etoposide. By Western blot, MVP was also detected in all tumor samples taken from 7 glioma and 3 meningioma patients. Taken together, these data suggest overexpression of MVP as one explanation for the low efficacy of chemotherapeutic treatment of astrocytic brain tumors.

Fibroblast Growth Factor Receptors as Therapeutic Targets in Human Melanoma: Synergism with BRAF Inhibition

Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1-4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.

FGF5 as an oncogenic factor in human glioblastoma multiforme: autocrine and paracrine activities

Fibroblast growth factor 5 (FGF5) is widely expressed in embryonic but scarcely in adult tissues. Here we report simultaneous overexpression of FGF5 and its predominant high-affinity receptor (FGFR1 IIIc) in astrocytic brain tumour specimens (N=49) and cell cultures (N=49). The levels of both ligand and receptor increased with enhanced malignancy in vivo and in vitro. Furthermore, secreted FGF5 protein was generally present in the supernatants of glioblastoma (GBM) cells. siRNA-mediated FGF5 downmodulation reduced moderately but significantly GBM cell proliferation while recombinant FGF5 (rFGF5) increased this parameter preferentially in cell lines with low endogenous expression levels. Apoptosis induction by prolonged serum starvation was significantly prevented by rFGF5. Moreover, tumour cell migration was distinctly stimulated by rFGF5 but attenuated by FGF5 siRNA. Blockade of FGFR1-mediated signals by pharmacological FGFR inhibitors or a dominant-negative FGFR1 IIIc protein inhibited GBM cell proliferation and/or induced apoptotic cell death. Moreover, rFGF5 and supernatants of highly FGF5-positive GBM cell lines specifically stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells. In summary, we demonstrate for the first time that FGF5 contributes to the malignant progression of human astrocytic brain tumours by both autocrine and paracrine effects.

Galectin-1 Is Implicated in the Protein Kinase C ε/Vimentin-Controlled Trafficking of Integrin-β1 in Glioblastoma Cells

Cell motility and resistance to apoptosis characterize glioblastoma (GBM) growth and malignancy. In our current work we report that galectin‐1, a homodimeric adhesion molecule and carbohydrate‐binding protein with affinity for β‐galactosides, is linked with cell surface expression of integrin β1 and the process of integrin trafficking. Using immunofluorescence, depletion of galectin‐1 through both stable knockdown and transient‐targeted small interfering RNA (siRNA) treatment induces an intracellular accumulation of integrin‐β1 coincident with a diminution of integrin‐β1 at points of cellular adhesion at the cell membrane. Galectin‐1 depletion does not alter the gene expression level of integrin‐β1. Transient galectin‐1 depletion effectuates as well the perinuclear accumulation of protein kinase C epsilon (PKCε) and the intermediate filament vimentin, both of which have been shown to mediate integrin recycling in motile cells. Our results argue for the involvement of galectin‐1 in the PKCε/vimentin‐controlled trafficking of integrin‐β1. The understanding of molecular mediators such as galectin‐1 and the pathways through which they drive the cell invasion so descriptive of GBM is anticipated to reveal potential therapeutic targets that promote glioma malignancy.

Fibroblast growth factor receptor 3-IIIc mediates colorectal cancer growth and migration

Background:Deregulation of fibroblast growth factor receptor 3 (FGFR3) is involved in several malignancies. Its role in colorectal cancer has not been assessed before.Methods:Expression of FGFR3 in human colorectal tumour specimens was analysed using splice variant-specific real-time reverse transcriptase PCR assays. To analyse the impact of FGFR3-IIIc expression on tumour cell biology, colon cancer cell models overexpressing wild-type (WT-3b and WT3c) or dominant-negative FGFR3 variants (KD3c and KD3b) were generated by either plasmid transfection or adenoviral transduction.Results:Although FGFR3 mRNA expression is downregulated in colorectal cancer, alterations mainly affected the FGFR3-IIIb splice variant, resulting in an increased IIIc/IIIb ratio predominantly in a subgroup of advanced tumours. Overexpression of WT3c increased proliferation, survival and colony formation in all colon cancer cell models tested, whereas WT3b had little activity. In addition, it conferred sensitivity to autocrine FGF18-mediated growth and migration signals in SW480 cells with low endogenous FGFR3-IIIc expression. Disruption of FGFR3-IIIc-dependent signalling by dominant-negative FGFR3-IIIc or small interfering RNA-mediated FGFR3-IIIc knockdown resulted in inhibition of cell growth and induction of apoptosis, which could not be observed when FGFR3-IIIb was blocked. In addition, KD3c expression blocked colony formation and migration and distinctly attenuated tumour growth in SCID mouse xenograft models.Conclusion:Our data show that FGFR3-IIIc exerts oncogenic functions by mediating FGF18 effects in colorectal cancer and may constitute a promising new target for therapeutic interventions.

Prognostic significance of telomerase-associated parameters in glioblastoma: effect of patient age

BACKGROUND Glioblastoma multiforme (GBM) is a heterogeneous, highly aggressive primary brain tumor with strongly variable patient survival. Because reliable prognostic biomarkers are lacking, we investigated the relation between telomerase-associated parameters and the disease course. METHODS Telomerase-associated parameters were determined in 100 GBM tissues and associated with clinical characteristics and overall survival. Expressions of telomere length, telomerase activity (TA), and human telomerase reverse transcriptase (hTERT) were analyzed by quantitative PCR, telomeric repeat amplification protocol assay, and reverse transcriptase-PCR, respectively. Mutation status of isocitrate dehydrogenase (IDH)1 was determined by direct sequencing, and O(6)-methylguanine DNA methyltransferase (MGMT) promoter methylation by methylation-specific PCR. RESULTS Of 100 GBM tissues, 61 were positive for both hTERT mRNA and TA, with a highly significant correlation between both parameters (linear regression, P < .0001). Telomere length determination revealed a significant difference between the hTERT/TA-positive and -negative subgroups, with markedly longer telomeres in the hTERT/TA-negative cohort (unpaired Students t-test, P = .0001). Accordingly, significantly shorter telomeres were detected in GBM tissues derived from older patients (>60 y at diagnosis, P < .0001). While no association of telomere parameters with MGMT promoter status was found, all tumors with IDH1 mutation (6/100) were negative for both hTERT expression and TA and harbored significantly longer telomeres. Patients with tumors lacking hTERT expression/TA showed a significant survival benefit (Kaplan-Meier test, both P < .01), which, however, was based exclusively on the younger patient subgroup (≤60 y, both P < .005; >60 y, both ns). CONCLUSIONS Telomerase activation is not an independent prognostic parameter in GBM but predicts aggressive tumor behavior solely in a younger patient cohort.