Influence of aflibercept on platelet activation profile.

Aflibercept appears to accumulate in systemic circulation following intravitreal injections in therapy of neovascular age-related macular degeneration. This gives raise to the question of whether aflibercept affects platelets and their function such as activation and aggregation, which are substantial in the pathogenesis of an arterial thromboembolic event (ATE). In order to determine the effect of aflibercept in platelet activation, platelets from healthy volunteers were treated with aflibercept and its solvents at equal concentrations (0.04 µg/mL - 4 µg/mL - 40 µg/mL - 400 µg/mL - 4 mg/mL) for 10 and 30 min before addition of agonists. IgG1 antibody was used as a control. The surface expression of GPIIb/IIIa, P-selectin, and platelet-bound stromal-cell-derived factor-1, which are potential blood biomarkers for ATEs, was determined on resting and activated platelets by the multispectral imaging flow cytometry, combining the features of flow cytometry with fluorescence microscopy. Platelet aggregation was assessed with light transmission aggregometry. To determine whether aflibercept directly interacts with platelets, aflibercept was labeled with the fluorescence FITC. Co-treatment of platelets with thrombin or PAR-4-AP and aflibercept resulted in increased activation of the fibrinogen receptor GPIIb/IIIa in comparison to controls (P < 0.05). Interestingly, the expression of platelet-derived P-selectin and SDF-1 was not affected by aflibercept, except thrombin-activated CD62P with 0.04 µg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.54, IC = 1.201-1.879 vs. MSI = 1.37, IC = 1.136-1.604 [P = 0.031]) and SDF-1 with 4 mg/mL aflibercept (aflibercept vs. its solvent: MSI = 1.971, IC = 1.206-2.737 vs. MSI = 1.200, IC = 0.738-1.662 [P = 0.041]). Although the levels of platelet-bound aflibercept-FITC were significantly increased in all activated platelets, no effect was observed in platelet aggregation. Albeit no impact of aflibercept was found on platelet aggregation under the studied experimental conditions, the increased activation of the fibrinogen receptor GPIIb/IIIa and the presence of a direct interaction between aflibercept and platelets may partially explain the risk of ATE in patients under aflibercept treatment due to FcγRIIa mediated αIIbß3 outside-in integrin signaling and transport of aflibercept into platelets. Therefore, the Fc domain seems to be involved in interactions between aflibercept and platelets. Further research is needed to explain the role of Fc containing aflibercept in the pathogenesis of drug-associated vascular events involving platelets, coagulation cascade, extracellular matrix proteins and other cells.

The clinical relevance of birch pollen profilin cross-reactivity in sensitized patients.

BACKGROUND: Overlapping seasons and cross-reactivity, especially to grass pollen profilin, can hamper the diagnosis of birch pollen allergy. To identify the primary sensitizing allergen and the clinical relevance of cross-sensitization, we correlated sensitization profiles with in vitro and in vivo tests, symptom scores, and pollen counts. METHODS: A total of 433 patients with positive skin prick test (SPT) to birch pollen were analyzed regarding IgE to major birch and grass pollen allergens Bet v 1 and Phl p 1/p 5 and the profilins Bet v 2 and Phl p 12. Subgroups were analyzed by basophil activation test (BAT) and CAP-FEIA-based cross- and self-inhibition tests. RESULTS: A total of 349 patients were sensitized to Bet v 1, 44 patients to both Bet v 1 and Bet v 2, and 15 patients to Bet v 2 only. From Bet v 2-sensitized patients, 40 were also sensitized to Phl p 12. Ex vivo, Bet v 2 and Phl p 12 induced dose-dependent activation in basophils of these patients. Cross- and self-inhibition tests with both allergens confirmed cross-reactivity. However, semiquantitative analysis of SPTs demonstrated markedly increased reactivity to grass compared to birch pollen extract in Bet v 2 only sensitized patients. Accordingly, in most of those patients, clinical symptoms precisely correlated with grass pollen counts. CONCLUSION: Identification of the clinically relevant and sensitizing allergen needs correlation of actual pollen counts with clinical symptoms and sensitization status to major allergens. Semiquantitative analysis of SPT or BAT and determining profilin-specific IgE can contribute to making the diagnosis.

Ezrin-dependent promotion of glioma cell clonogenicity, motility, and invasion mediated by BCL-2 and transforming growth factor-beta2.

Ezrin belongs to the ezrin-radixin-moesin family proteins, which cross-link actin cytoskeleton and plasma membrane. Malignant glioma cells are paradigmatic for their strong migratory and invasive properties. Here, we report that the expression of dominant-negative ezrins inhibits clonogenicity, migration, and invasiveness of human malignant glioma cells. Furthermore, dominant-negative ezrins block hepatocyte growth factor (HGF)-mediated stimulation of clonogenicity and migration, without altering HGF-induced protein kinase B/Akt and focal adhesion kinase phosphorylation. Glioma cells expressing dominant-negative ezrins exhibit a shift of the BCL-2/BAX rheostat toward apoptosis, reduced alpha(V)beta(3) integrin expression and reduced matrix metalloproteinase (MMP) expression and activity. These changes are associated with a dramatic loss of transforming growth factor beta(2) (TGF-beta(2)) release. Exogenous supplementation of TGF-beta(2) overcomes the inhibitory effects of dominant-negative ezrins on migration and clonogenicity. A neutralizing TGF-beta(2) antibody mimics the effects of dominant-negative ezrins on clonogenicity and migration. Exogenous HGF markedly induces TGF-beta(2) protein levels, and a neutralizing TGF-beta(2) antibody abolishes the HGF-mediated increase in glioma cell motility. Finally, TGF-beta(2) does not modulate BCL-2 or BAX expression, but BCL-2 gene transfer increases the levels of latent and active TGF-beta(2). Intracranial xenografts of U87MG glioma cells transfected with the dominant-negative ezrins in athymic mice grow to significantly smaller volumes, and the median survival of these mice is 50 d compared with 28 d in the control group. These data define a novel pathway for HGF-induced glioma cell migration and invasion, which requires ezrin, changes in the BCL-2/BAX rheostat, and the induction of TGF-beta(2) expression in vitro, and underscore the important role of HGF signaling in vivo.

Secreted Frizzled-related proteins inhibit motility and promote growth of human malignant glioma cells.

Cellular resistance to multiple proapoptotic stimuli and invasion of surrounding brain tissue by migrating tumor cells are main obstacles to an effective therapy for human malignant glioma. Here, we report that the Wnt family of embryonic differentiation genes modulate growth of malignant glioma cells in vitro and in vivo and inhibit cellular migration in vitro. sFRPs (soluble Frizzled-related proteins) are soluble proteins that bind to Wnt and interfere with Wnt signaling. We find that sFRP-1 and sFRP-2 are produced by the majority of longterm and ex vivo malignant glioma cell lines. Glioma cells that ectopically express sFRPs exhibit increased clonogenicity and enhanced resistance to serum starvation. In contrast, sFRPs do not modulate glioma cell susceptibility to apoptosis induced by the cytotoxic cytokines, CD95 (Fas/APO-1) ligand (CD95L) or Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL), or various cytotoxic drugs. sFRP-2 strongly promotes the growth of intracranial glioma xenografts in nude mice. In contrast, enhanced expression of sFRPs inhibits the motility of glioma cells in vitro. sFRP-mediated effects on glioma cells are accompanied by decreased expression and activity of matrix metalloproteinase-2 (MMP-2) and decreased tyrosine phosphorylation of beta-catenin. Thus, sFRPs promote survival under non-supportive conditions and inhibit the migration of glioma cells. We suggest that the regulation of these cellular processes involves expression of MMP-2 and tyrosine phosphorylation of beta-catenin. These data support a function for Wnt signaling and its modulation by sFRPs in the biology of human gliomas. Oncogene (2000) 19, 4210 - 4220

Bag-1 and Bcl-2 gene transfer in malignant glioma: modulation of cell cycle regulation and apoptosis.

Bag-1 is a heat shock 70 kDa (Hsp70)-binding protein that can collaborate with Bcl-2 in suppressing apoptosis under some conditions. Here, we report that 11 of 12 human glioma cell lines express Bag-1 protein in vitro. Moreover, 15 of 19 human glioblastomas expressed Bag-1 as assessed by immunohistochemistry in primary tumor specimens. To examine the biological effects of Bag-1 in glioma cells, we expressed Bag-1 or Bcl-2 transgenes in 2 human malignant glioma cell lines, LN-18 and LN-229. Bag-1 significantly slowed glioma cell growth and reduced clonogenicity of both cell lines in vitro. Coexpressed Bcl-2 abrogated these effects of Bag-1. Intracranial LN-229 glioma xenografts implanted into nude mice revealed a substantial growth advantage afforded by Bcl-2. Bag-1 had no such effect, either in the absence or presence of Bcl-2. Upon serum starvation in vitro, Bcl-2 prevented cell death whereas Bag-1 did not. Both Bcl-2 and Bag-1 slowed proliferation of serum-starved cells when expressed alone. Importantly, coexpression of Bcl-2 and Bag-1 provided a distinct growth advantage under conditions of serum starvation that is probably the result of (i) the death-preventing activity of Bcl-2 and (ii) the property of Bag-1 to overcome a Bcl-2-mediated enhancement of exit from the cell cycle. In contrast to these Bcl-2/Bag-1 interactions observed under serum starvation conditions, Bag-1 did not further enhance the strong protection from staurosporine-, CD95 (Fas/Apo1) ligand-, Apo2 ligand (TRAIL)- or chemotherapeutic drug-induced apoptosis afforded by Bcl-2. Taken together, these results indicate a role for Bag-1/Bcl-2 interactions in providing a survival advantage to cancer cells in a deprived microenvironment that may be characteristic of ischemic/hypoxic tumors such as human glioblastoma multiforme, and suggest that Bcl-2/Bag-1 interactions also modulate cell proliferation.

Betulinic acid-induced apoptosis in glioma cells: A sequential requirement for new protein synthesis, formation of reactive oxygen species, and caspase processing.

Betulinic acid (BA), a pentacyclic triterpene, is an experimental cytotoxic agent for malignant melanoma. Here, we show that BA triggers apoptosis in five human glioma cell lines. BA-induced apoptosis requires new protein, but not RNA, synthesis, is independent of p53, and results in p21 protein accumulation in the absence of a cell cycle arrest. BA-induced apoptosis involves the activation of caspases that cleave poly(ADP ribose)polymerase. Interactions of death ligand/receptor pairs of the CD95/CD95 ligand family do not mediate BA-induced caspase activation. BA enhances the levels of BAX and BCL-2 proteins but does not alter the levels of BCL-xS or BCL-xL. Ectopic expression of BCL-2 prevents BA-induced caspase activation, DNA fragmentation, and cell death. Furthermore, BA induces the formation of reactive oxygen species that are essential for BA-triggered cell death. The generation of reactive oxygen species is blocked by BCL-2 and requires new protein synthesis but is unaffected by caspase inhibitors, suggesting that BA toxicity sequentially involves new protein synthesis, formation of reactive oxygen species, and activation of crm-A-insensitive caspases.

TGF-beta-independent induction of immunogenicity by decorin gene transfer in human malignant glioma cells.

Ectopic expression of the proteoglycan, decorin, abrogates the growth of experimental C6 gliomas in the rat. Since gliomas release large amounts of transforming growth factor-beta (TGF-beta) and since decorin is a TGF-beta antagonist, decorin gene transfer-mediated abrogation of glioma growth in vivo may involve enhanced immunogenicity of the tumor cells. Here, we report that human glioma cells stimulate alloreactive immune responses when engineered to express decorin whereas parental glioma cells are non-immunogenic in vitro. The alloreactive immune response is mediated by CD8+ and CD4+ T cells as well as by NK cells. The immunosuppression exerted by parental or mock-transfected glioma cells is mediated by soluble factors and can in part be mimicked by exogenous TGF-beta. However, neutralizing anti-TGF-beta antibodies do not reverse glioma-mediated immunosuppression, suggesting that decorin abrogates glioma-induced immune cell inhibition by interfering with the activity of other, so far unidentified glioma-secreted mediators. We conclude that enhanced immunogenicity may mediate the antineoplastic effects of decorin gene therapy for malignant glioma but that factors other than TGF-beta may be responsible for glioma-induced immunosuppression.

Predicting chemoresistance in human malignant glioma cells: the role of molecular genetic analyses.

Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we asked whether alterations in the p53 and RB pathways and the expression of six BCL-2 family proteins predicted acute cytotoxicity and clonogenic cell death induced by BCNU, vincristine, cytarabine, teniposide, doxorubicin, camptothecin or beta-lapachone in 12 human malignant glioma cell lines. Neither wild-type p53 status, nor p53 protein accumulation, nor p21 or MDM-2 levels, nor differential expression of BCL-2 family proteins predicted drug sensitivity, except for an association of BAX with higher beta-lapachone sensitivity in acute cytotoxicity assays. p16 protein expression was associated with high doubling time and chemoresistance. We conclude that some important molecular changes, which are involved in the development of gliomas and attributed a role in regulating vulnerability to apoptosis, may not determine the response to chemotherapy in these tumors.

Taxol-mediated augmentation of CD95 ligand-induced apoptosis of human malignant glioma cells: association with bcl-2 phosphorylation but neither activation of p53 nor G2/M cell cycle arrest.

The anti-tumour alkaloid taxol shows strong cytotoxic and antiproliferative activity in two human malignant glioma cell lines, T98G and LN-229. CD95 (Fas/APO-1) ligand is a novel cytotoxic cytokine of the tumour necrosis factor (TNF) family that exerts prominent antiglioma activity. At clinically relevant taxol concentrations of 5-100 nM, taxol and CD95 ligand showed significant synergistic cytotoxicity and growth inhibition. High concentrations of taxol induced G/M cell cycle arrest in both cell lines. The synergy of taxol and CD95 ligand was independent of cell cycle effects of taxol as synergy was achieved at much lower taxol concentrations than G2/M arrest and as cell cycle effects of taxol were unaffected by co-exposure to CD95 ligand. Similarly, high concentrations of taxol were required to induce p53 activity in the p53 wild-type cell line LN-229. This effect was not modulated by CD95 ligand, suggesting that synergy is also independent of p53 activation. However, taxol induced a mobility shift of the bcl-2 protein on immunoblot analysis, indicative of bcl-2 phosphorylation. Bcl-2 phosphorylation on serine was confirmed by immunoprecipitation and phosphoserine immunoblot analysis. Considering (1) that phosphorylation of bcl-2 interferes with its heterodimerization with bax and (2) the inhibition of CD95-mediated apoptosis by bcl-2, we propose that taxol sensitizes malignant glioma cells to CD95 ligand by increasing the functional bax/bcl-2 rheostat in favour of bax and thus cell death.

Hypericin-induced apoptosis of human malignant glioma cells is light-dependent, independent of bcl-2 expression, and does not require wild-type p53.

Hypericin and tamoxifen are experimental agents for the adjuvant chemotherapy of malignant glioma. We report that hypericin and tamoxifen induce apoptosis of 7 human malignant glioma cell lines in a concentration- and time-dependent manner. Illumination is essential for the cytotoxicity of hypericin but not tamoxifen. Apoptosis is unaffected by inhibitors of RNA and protein synthesis or free radical scavengers, does not require wild-type p53 activity, and occurs in glioma cells expressing high levels of bcl-2. There is no correlation between hypericin and tamoxifen-induced cytotoxicity and inhibition of protein kinase C (PKC). Ectopic expression of a murine bcl-2 transgene provides modest protection from tamoxifen but does not affect hypericin toxicity. Hypericin and tamoxifen do not modulate glioma cell killing induced by tumor necrosis factor-alpha (TNF-alpha) or CD95 ligand. Both drugs augment the acute cytotoxicity of various cancer chemotherapy drugs but fail to shift their EC50 values in modified colony formation assays. These data do not provide further supportive evidence how to enhance the limited efficacy of tamoxifen treatment for human malignant glioma. However, hypericin is a promising agent for the treatment of malignant glioma if local photodynamic activation of hypericin in the glioma tissue can be achieved.